Glucoamylase variants

ABSTRACT

The invention relates to a variant of a parent fungal glucoamylase, which exhibits improved thermal stability and/or increased specific activity using saccharide substrates.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser.No. 09/351,814, filed on Jul. 12, 2001, and claims priority under 35U.S.C. 119 of Danish application nos. PA 1998 00937 and PA 19980167filed on Jul. 15, 1998 and Dec. 17, 1998, respectively, and U.Sprovisional application Nos. 60/093,528 and 60/115,545 filed on Jul. 21,1998 and Jan. 12, 1999, respectively, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to novel glucoamylase variants(mutants) of parent AMG, in particular with improved thermal stabilityand/or increased specific activity suitable for, e.g., starchconversion, e.g., for producing glucose from starch. More specifically,the present invention relates to glucoamylase enzyme variants and theuse of such variant enzymes.

BACKGROUND OF THE INVENTION

[0003] Glucoamylase (1,4-_(α)-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeasts, withthose from Aspergillus being commercially most important.

[0004] Commercially, the glucoamylase enzyme is used to convert cornstarch which is already partially hydrolyzed by an _(α)-amylase toglucose. The glucose is further converted by glucose isomerase to amixture composed almost equally of glucose and fructose. This mixture,or the mixture further enriched with fructose, is the commonly used highfructose corn syrup commercialized throughout the world. This syrup isthe world's largest tonnage product produced by an enzymatic process.The three enzymes involved in the conversion of starch to fructose areamong the most important industrial enzymes produced.

[0005] One of the main problems that exist with regard to the commercialuse of glucoamylase in the production of high fructose corn syrup is therelatively low thermal stability of glucoamylase. Glucoamylase is not asthermally stable as _(α)-amylase or glucose isomerase and it is mostactive and stable at lower pH's than either _(α)-amylase or glucoseisomerase. Accordingly, it must be used in a separate vessel at a lowertemperature and pH.

[0006] Glucoamylase from Aspergillus niger has a catalytic (aa 1-440)and a starch binding domain (aa 509-616) separated by a long and highlyO-glycosylated linker (Svensson et al. (1983), Carlsberg Res. Commun.48, 529-544, 1983 and (1986), Eur. J. Biochem. 154, 497-502). Thecatalytic domain (aa 1-471) of glucoamylase from A. awamori var. X100adopt an (_(α)/_(α))₆-fold in which six conserved _(α→α) loop segmentsconnect the outer and inner barrels (Aleshin et al. (1992), J.Biol.Chem. 267, 19291-19298). Crystal structures of glucoamylase incomplex with 1-deoxynojirimycin (Harris et al. (1993), Biochemistry, 32,1618-1626) and the pseudotetrasaccharide inhibitors acarbose andD-gluco-dihydroacarbose (Aleshin et al. (1996), Biochemistry 35,8319-8328) furthermore are compatible with glutamic acids 179 and 400acting as general acid and base, respectively. The crucial role of theseresidues during catalysis have also been studied using proteinengineering (Sierks et al. (1990), Protein Engng. 3, 193-198; Frandsenet al. (1994), Biochemistry, 33, 13808-13816). Glucoamylase-carbohydrateinteractions at four glycosyl residue binding subsites, −1, +1, +2, and+3 are highlighted in glucoamylase-complex structures (Aleshin et al.(1996), Biochemistry 35, 8319-8328) and residues important for bindingand catalysis have been extensively investigated using site-directedmutants coupled with kinetic analysis (Sierks et al. (1989), ProteinEngng. 2, 621-625; Sierks et al. (1990), Protein Engng. 3, 193-198;Berland et al. (1995), Biochemistry, 34, 10153-10161; Frandsen et al.(1995), Biochemistry, 34, 10162-10169.

[0007] Different substitutions in A. niger glucoamylase to enhance thethermal stability have been described: i) substitution of c-helicalglycines: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505);ii) elimination of the fragile Asp-X peptide bonds, D257E and D293E/Q(Chen et al. (1995), Prot. Engng. 8, 575-582); prevention of deamidationin N182 (Chen et al. (1994), Biochem. J. 301, 275-281); iv) engineeringof additional disulphide bond, A246C (Fierobe et al. (1996),Biochemistry, 35, 8698-8704; and v) introduction of Pro residues inposition A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204.Furthermore Clark Ford presented a paper on Oct. 17, 1997, ENZYMEENGINEERING 14, Beijing/China Oct. 12-17, 1997, Abstract number:Abstract book p. 0-61. The abstract suggests mutations in positionsG137A, N20C/A27C, and S30P in a (not disclosed) Aspergillus awamoriglucoamylase to improve the thermal stability.

[0008] Additional information concerning glucoamylase can be found on anInternet homepage(http://www.public.iastate.edu/˜pedro/glase/glase.html) “GlucoamylaseWWW page” (Last changed Oct. 8, 1997) by Pedro M. Coutinho disclosesinformations concerning glucoamylases, including glucoamylases derivablefrom Aspergillus strains. Chemical and site-directed modifications inthe Aspergillus niger glucoamylase are listed.

BRIEF DISCLOSURE OF THE INVENTION

[0009] The object of the present invention is to provide improvedglucoamylase variants with improved thermostablility and/or increasedspecific activity suitable for use in, e.g., the saccharification stepin starch conversion processes.

[0010] The term “a glucoamylase variant with improved thermostability”means in the context of the present invention a glucoamylase variantwhich has a higher T_(½) (half-time) than the corresponding parentglucoamylase. The determination of T_(½) (Method I and Method II) isdescribed below in the “Materials & Methods” section.

[0011] The term “a glucoamylase variant with increased specificactivity” means in the context of the present invention a glucoamylasevariant with increased specific activity towards the _(α)-1,4 linkagesin the saccharide in question. The specific activity is determined ask_(cat) or AGU/mg (measured as described below in the “Materials &Methods” section). An increased specific activity means that the k_(cat)or AGU/mg values are higher when compared to the k_(cat) or AGU/mgvalues, respectively, of the corresponding parent glucoamylase.

[0012] The inventors of the present invention have provided a number ofimproved variants of a parent glucoamylase with improved thermostabilityand/or increased specific activity in comparison to the parentcorresponding enzyme. The improved thermal stability is obtained bysubstituting selected positions in a parent glucoamylase. This will bedescribed in details below.

[0013] Nomenclature

[0014] In the present description and claims, the conventionalone-letter and three-letter codes for amino acid residues are used. Forease of reference, glucoamylase variants of the invention are describedby use of the following nomenclature:

[0015] Original amino acid(s):position(s):substituted amino acid(s)

[0016] According to this nomenclature, for instance the substitution ofalanine for asparagine in position 30 is shown as:

[0017] Ala30Asn or A30N

[0018] a deletion of alanine in the same position is shown as:

[0019] Ala30* or A30*

[0020] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0021] Ala30AlaLys or A30AK

[0022] A deletion of a consecutive stretch of amino acid residues, suchas amino acid residues 30-33, is indicated as (30-33)* or _(Δ)(A30-N33).

[0023] Where a specific glucoamylase contains a “deletion” in comparisonwith other glucoamylases and an insertion is made in such a positionthis is indicated as:

[0024] *36Asp or *36D

[0025] for insertion of an aspartic acid in position 36

[0026] Multiple mutations are separated by plus signs, i.e.:

[0027] Ala30Asp+Glu34Ser or A30N+E34S

[0028] representing mutations in positions 30 and 34 substitutingalanine and glutamic acid for asparagine and serine, respectively.Multiple mutation may also be separated as follows, i.e., meaning thesame as the plus sign:

[0029] Ala30Asp/Glu34Ser or A30N/E34S

[0030] When one or more alternative amino acid residues may be insertedin a given position it is indicated as

[0031] A30N,E or A30N/E, or A30N or A30E

[0032] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

[0033] R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

BRIEF DESCRIPTION OF THE DRAWING

[0034]FIG. 1 shows the plasmid pCAMG91 containing the Aspergillus nigerG1 glucoamylase gene.

DETAILED DISCLOSURE OF THE INVENTION

[0035] A goal of the work underlying the present invention was toimprove the thermal stability and/or increase the specific activity ofparticular glucoamylases which are obtainable from fungal organisms, inparticular strains of the Aspergillus genus and which themselves hadbeen selected on the basis of their suitable properties in starchconversion or alcohol fermentation.

[0036] Identifying Positions and/or Regions to be Mutated to ObtainImproved Thermostability and/or Increased Specific Activity

[0037] Molecular dynamics (MD) simulations indicate the mobility of theamino acids in the protein structure (see McCammon, J A and Harvey, SC., (1987), “Dynamics of proteins and nucleic acids”. CambridgeUniversity Press.). Such protein dynamics are often compared to thecrystallographic B-factors (see Stout, G H and Jensen, L H, (1989),“X-ray structure determination”, Wiley). By running the MD simulation atdifferent protonation states of the titrate able residues, the pHrelated mobility of residues are simulated. Regions having the highestmobility or flexibility (here isotropic fluctuations) are selected forrandom mutagenesis. It is here understood that the high mobility foundin certain areas of the protein, can be thermally improved bysubstituting residues in these residues. The substitutions are directedagainst residues that will change the dynamic behaviour of the residuesto e.g. bigger side-chains and/or residues which have capability offorming improved contacts to residues in the near environment. The AMGfrom Aspergillus niger was used for the MD simulation. How to carry outMD simulation is described in the “Materials & Methods” section below.)

[0038] Regions found by Molecular dynamics (MD) simulations to besuitable for mutation when wanting to obtain improved thermal stabilityand/or increased specific activity are the following:

[0039] Region: 1-18,

[0040] Region: 19-35,

[0041] Region: 73-80,

[0042] Region: 200-212,

[0043] Region: 234-246,

[0044] Region: 334-341,

[0045] Region: 353-374,

[0046] Region: 407-411,

[0047] Region: 445-470,

[0048] Regions found to be of interest for increasing the specificactivity and/or improved thermostability are the regions in proximity tothe active site. Regions positioned in between the _(α)-helixes, andwhich may include positions on each side of the N- and C-terminal of the_(α)-helixes, at the substrate binding site is of importance for theactivity of the enzyme. These regions constitute the following regions:

[0049] Region: 40-62,

[0050] Region: 93-127,

[0051] Region: 170-184,

[0052] Region: 234-246,

[0053] Region: 287-319,

[0054] Region: 388-414.

[0055] Rhizopus, Talaromyces, such as Talaromyces emersonii (dislosed inWO 99/28448), and Thielavia have high specific activity towardsmaltodextrins, including maltose and maltohepatose. Therefore, regionsbeing of special interest regarding (transferring) increased specificactivity are:

[0056] Region: 200-212,

[0057] Region: 287-300,

[0058] Region: 305-319.

[0059] The present inventors have found that it is in fact possible toimprove the thermal stability and/or to increase the specific activityof a parent glucoamylase by modification of one or more amino acidresidues of the amino acid sequence of the parent glucoamylase. Thepresent invention is based on this finding.

[0060] Accordingly, in a first aspect the present invention relates toan improved variant of a parent glucoamylase comprising one or moremutations in the regions and positions described further below.

[0061] Parent Glucoamylases

[0062] Parent glucoamylase contemplated according to the presentinvention include fungal glucoamylases, in particular fungalglucoamylases obtainable from an Aspergillus strain, such as anAspergillus niger or Aspergillus awamori glucoamylases and variants ormutants thereof, homologous glucoamylases, and further glucoamylasesbeing structurally and/or functionally similar to SEQ ID NO: 2.Specifically contemplated are the Aspergillus niger glucoamylases G1 andG2 disclosed in Boel et al. (1984), “Glucoamylases G1 and G2 fromAspergillus niger are synthesized from two different but closely relatedmRNAs”, EMBO J. 3 (5), p. 1097-1102,. The G2 glucoamylase is disclosedin SEQ ID NO: 2. The G1 glucoamylase is disclosed in SEQ ID NO: 13.Another AMG backbone contemplated is Talaromyces emersonii, especiallyTalaromyces emersonii DSM disclosed in WO 99/28448 (Novo Nordisk).

[0063] Commercially Available Parent Glucoamylases

[0064] Commercially available parent glucoamylases include AMG from NovoNordisk, and also glucoamylase from the companies Genencor, Inc. USA,and Gist-Brocades, Delft, The Netherlands.

[0065] Glucoamylase Variants

[0066] In the first aspect the invention relates to a variant of aparent glucoamylase comprising one or more mutation(s) in the followingpositions(s) or region(s) in the amino acid sequence shown in NO: 2:

[0067] Region: 1-18,

[0068] Region: 19-35,

[0069] Region: 40-62,

[0070] Region: 73-80,

[0071] Region: 93-127,

[0072] Region: 170-184,

[0073] Region: 200-212,

[0074] Region: 234-246,

[0075] Region: 287-319,

[0076] Region: 334-341,

[0077] Region: 353-374,

[0078] Region: 388-414,

[0079] Region: 445-470,

[0080] and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: N20C, A27C, S30P, Y48W, Y50F, W52F, R54K/L, D55G/V, G57A,K108R, D112Y, Y116A/W, S119C/W/E/G/Y/P, W120H/L/F/Y, G121T/A, R122Y,P123G, Q124H, R125K, W170F, N171S, Q172N, T173G, G174C, Y175F, D176N/E,L177H/D, W178R/D, E179Q/D, E180D/Q, V181D/A/T, N182A/D/Q/Y/S, G183K,S184H, W212F, R241K, A246C, D293E/Q, A302V, R305K, Y306F, D309N/E,Y312W, W317F, E389D/Q, H391W, A392D, A393P, N395Q, G396S, E400Q/C,Q401E, G407D, E408P, L410F, S411A/G/C/H/D, S460P

[0081] In an embodiment the region mutated is the Region: 1-18.

[0082] Specific preferred positions contemplated include one or more of:

[0083] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.

[0084] Specific mutations include one or more of: A1V, T2P/Q/R/H/M/E/K,L3N, N9A, A11E/P, I18V.

[0085] Preferred combinations of mutations include one or more of:

[0086] A1V+L66R+Y402F+N427S+S486G,

[0087] T2K+S30P+N427M+S44G+V470M,

[0088] T2E+T379A+S386K+A393R,

[0089] T2Q+A11P+S394R,

[0090] T2R+L66V+S394P+Y402F

[0091] T2M+N9A+T390R+D406N+L410R,

[0092] T2R+S386R+A393R,

[0093] A11P+T2Q+S394R,

[0094] A11E+E408R,

[0095] I18V+T51S+S56A+V59T+L60A.

[0096] In an embodiment the region mutated is the Region: 19-35.

[0097] Preferred sub-regions include one or more of: 21-26, 31-35.

[0098] Specific preferred positions contemplated include one or more of:19, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35.

[0099] Specific mutations include one or more of: L19N, N20T, G23A,A24S/T, D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V, D33R/K/H,S34N.

[0100] In an embodiment the region mutated is the Region: 40-62.

[0101] Preferred sub-regions include one or more of: 40-47, 58-62.

[0102] Specific preferred positions contemplated include one or more of:40, 41, 42, 43, 44, 45, 46, 47, 49, 51, 53, 56, 58, 59, 60, 61, 62.

[0103] Specific mutations include one or more of:

[0104] S40C/A/G,

[0105] T43R,

[0106] T51S/D,

[0107] T53D,

[0108] S56A/C,

[0109] V59T/A,

[0110] L60A.

[0111] Preferred combinations of mutations include one or more of:

[0112] T51 S+S56A+V59T+L60A+I18V

[0113] V59A+A393R+T490A+PLASD(N-terminal extension).

[0114] In an embodiment the region mutated is the Region: 73-80.

[0115] Specific preferred positions contemplated include one or more of:

[0116] 73, 74, 75, 76, 77, 78, 79, 80.

[0117] Specific mutations include one or more of:

[0118] S73P/D/T/N/Q/E,

[0119] L74I/D,

[0120] L75A/R/N/D/C/Q/E/G/H/I/K/M/P/S/T/V, preferred are I/N/D,

[0121] S76A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred T/A)

[0122] T77V/T,

[0123] 178V,

[0124] E79A/R/N/D/C/Q/G/H/I/L/K/F/M/P/S/T/Y/V, preferred are Q/R/K)

[0125] N80A/R/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/N, preferred are

[0126] H/D/E/R/K/T/S/Y.

[0127] In an embodiment the region mutated is the Region: 93-127.

[0128] In an additional embodiment the sub-region is: Region: 93-124.

[0129] Preferred sub-regions include one or more of: 93-107, 109-111,113-115.

[0130] Specific preferred positions contemplated include one or more of:93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 109, 110,111, 113, 114, 115, 117, 118, 126, 127.

[0131] Specific mutations include one or more of: N93T,

[0132] P94V,

[0133] S95N,

[0134] D97S,

[0135] L98S/P,

[0136] S100T/D,

[0137] A102S/*,

[0138] P107M/L/A/G/S/T/V/I,

[0139] N110T,

[0140] V111P,

[0141] D112N,

[0142] E113M/A,

[0143] T114S,

[0144] A115Q/A,

[0145] Y116F,

[0146] S119A/R/N/D/Q/H/I/L/K/F/M/T/V, preferred is A,

[0147] R122A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/V,

[0148] G127A.

[0149] Preferred combinations of mutations include one or more of:S119P+G447S,

[0150] S119P+Y402F,

[0151] S119P+A393R,

[0152] S119P+1189T+223F+F227Y+Y402F

[0153] S119P+T416H+Y402F+Y312Q.

[0154] In an embodiment the region mutated is the Region: 170-184.

[0155] Specific mutations include one or more of:

[0156] N171 R/K/Q/E/W/F/Y,

[0157] Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

[0158] T173K/R/S/N/Q,

[0159] G174A, S,

[0160] Y175N/Q/D/E,

[0161] D176L

[0162] L177I

[0163] E180N/M

[0164] V181I/T

[0165] N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V.

[0166] G183A

[0167] S184D/N/E/Q/L/I/T/R/K.

[0168] In an embodiment the region mutated is the Region: 200-212.

[0169] Preferred sub-regions include: 200-211.

[0170] Specific preferred positions contemplated include one or more of:

[0171] 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

[0172] Specific mutations include one or more of: A201D, , F202L, A203L,T204K, A205R/S, V206L/N, G207N, S208H/T/D, S209T, S211P, W212N/A/T.

[0173] In an embodiment the region mutated is the Region: 234-246.

[0174] Preferred sub-regions include one or more of: 234-240, 242-245.

[0175] Specific preferred positions contemplated include one or more of:

[0176] 234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

[0177] Specific mutations include one or more of:

[0178] L234AR/N/D/C/Q/E/G/H/I/K/M/F/P/S/T/W/Y/V.

[0179] A235S

[0180] F237Y/H/N/D.

[0181] D238T/S.

[0182] S239A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V.

[0183] S240G

[0184] S242S/P/T/A/Y/H/N/D.

[0185] G243S/P/T/A/Y/H/N/D.

[0186] K244R,

[0187] A246T.

[0188] Preferred combinations of mutations include one or more of:A246T+T721.

[0189] In an embodiment the region mutated is the Region: 287-319.

[0190] Preferred sub-regions include one or more of: 287-292, 294-301,313-316.

[0191] Specific preferred positions contemplated include one or more of:

[0192] 287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300,301,

[0193] 303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

[0194] Specific mutations include one or more of:

[0195] S287A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

[0196] I288L/N/Q,

[0197] Y289F,

[0198] T290A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/V,

[0199] L291I/D/N,

[0200] N292D,

[0201] D293A/R/N/C/Q/E/G/H/I/L/K/M/S/T/V,

[0202] G294A/R/N/D/C/Q/E/H/I/L/K/M/P/S/T/V,

[0203] L295A/R/N/D/C/Q/E/G/H/K/M/S/T/V,

[0204] S296A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

[0205] D297A/R/N/C/Q/E/G/H/I/L/K/M/P/S/T/V,

[0206] S298A/R/N/D/C/Q/E/G/H/I/L/K/F/M/T/V,

[0207] E299A/R/N/D/C/Q/G/H/I/L/K/M/S/T/V.

[0208] V301T/I,

[0209] A302R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V/, preferred S, V303T/I,

[0210] G304A,

[0211] R305A/N/D/C/Q/E/G/H/I/L/F/M/P/S/T/W/Y/V,

[0212] Y306A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/V.

[0213] E308A/R/N/D/C/Q/G/H/I/L/K/M/P/S/T/V, preferred Q,

[0214] D309L,

[0215] T310V/S,

[0216] Y311N,

[0217] Y312Q/N,

[0218] N313T/S/G

[0219] N315Q/E/R,

[0220] F318A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/Y/V, preferred is Y.

[0221] Preferred combinations of mutations include one or more of:

[0222] Y312Q+S119P+T416H+Y402F,

[0223] Y312Q+S119P+Y402F+T416H+S411V,

[0224] Y312Q+T416H

[0225] N313G+F318Y.

[0226] In an embodiment the region mutated is the Region: 334-341.

[0227] Specific preferred positions contemplated include one or more of:

[0228] 334, 335, 336, 337, 338, 339, 340, 341.

[0229] Specific mutations include one or more of:

[0230] D336A/R/N/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V.

[0231] K337A/R/N/D/C/Q/E/G/H/I/L/M/F/P/S/T/W/Y/V.

[0232] Q338A/R/N/D/C/G/H/I/L/F/P/S/T/W/Y/V.

[0233] G339S/P/T/A.

[0234] S340I/T/N/V/A/D/G.

[0235] L341F/L/I/V.

[0236] Preferred combinations of mutations include one or more of:

[0237] S340G+D357S+T360V+S386P.

[0238] In an embodiment the region mutated is the Region: 353-374.

[0239] Specific preferred positions contemplated include one or more of:

[0240] 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374.

[0241] Specific mutations include one or more of:

[0242] A353D/S,

[0243] S356P/N/D,

[0244] D357S,

[0245] A359S,

[0246] T360V,

[0247] G361S/P/T/A,

[0248] T362R,

[0249] S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0250] S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0251] S366T,

[0252] S368P/T/A,

[0253] T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0254] S371Y/H/N/D,

[0255] S372F/Y/C/P/H/R/I/T/N/S/V/A/D/G.

[0256] Preferred combinations of mutations include one or more of:

[0257] S356P+S366T,

[0258] D357S+T360V+S371H.

[0259] D357S+T360V+S386P+S340G.

[0260] In an embodiment the region mutated is the Region: 388-414.

[0261] Preferred sub-regions include one or more of: 397-399, 402-406,412-414.

[0262] Specific preferred positions contemplated include one or more of:

[0263] 388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409,412, 413, 414.

[0264] Specific mutations include one or more of: T390R,

[0265] A393R,

[0266] S394P/R,

[0267] M398L,

[0268] S399A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/T/W/Y/V, preferred are T/Q/C,

[0269] Y402A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/V, preferred is F,

[0270] D403S,

[0271] S405T,

[0272] D406N,

[0273] E408C/R,

[0274] A409R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred is P,

[0275] L401R/I,

[0276] S411R/N/Q/E/I/L/K/F/M/P/T/W/Y/V, preferred is V,

[0277] A412C,

[0278] R413A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V.

[0279] D414A.

[0280] Preferred combinations of mutations include one or more of:

[0281] A393R+T490A+V59A+PLASD(N-terminal extension)

[0282] S394R+T2Q+A11P,

[0283] Y402F+S411V,

[0284] Y402F+S411V+S119P

[0285] Y402F+S411V+S119P+A393R,

[0286] Y402F+Y312Q+S119P+T416H,

[0287] E408R+S386N,

[0288] E408R+A425T+S465P+T494A,

[0289] L410R+A393R,

[0290] Y402F+Y312Q+S119P+T416H+S411V+A393R.

[0291] In an embodiment the region mutated is the Region: 445-470.

[0292] Preferred sub-regions include one or more of: 445-459, 461-470.

[0293] Specific preferred positions contemplated include one or more of:

[0294] 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 461, 462, 463, 464, 465, 466, 467, 467, 468, 469, 470.

[0295] Specific mutations include one or more of: G447S, G456C/P, S465P.

[0296] Specific variants include variants having one or more of thefollowing substitutions: A1V, T2E/P/Q/R/H/M, L3P/N, N9A, A11P/E, I18V,L19N, N20T, G23A, A24S/T, D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A,A32V, D33R/K/H, S34N, S40C, T43R, T51D/S, T53D, S56A/C, V59T/A, L60A,N93T, P94V, S95N, D97S, L98P/S, S100T/D, A102S/*, N110T, V111P,D112N,E113M/A, T114S, A115Q/G, Y116F, S119A, G127A, N182E, A201D, F202L,A203L, T204K, A205R/S, V206L/N, G207N, S208H/T/D, S209T, S211P,W212N/A/T, A246T Y312Q, N313T/S/G, A353D/S, S356P/N/D, D357S, A359S,T360V, G361S/P/T/A, T362R, S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,S365A/R/N/D/C/Q/E/G/H/L/K/M/F/P/T/W/Y/V, S366T, S368P/T/A,T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S371Y/H/N/D,S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G, T390R, A393R, S394R/P, M398L,S399C/Q/T, Y402F, D403S, S405T, D406N,E408C/R, L410I/R, S411V, A412C,D414A, G447S, S465P.

[0297] Improved Thermal Stability

[0298] In a second aspect the invention relates to a variant of a parentglucoamylase with improved thermal stability, in particular in the rangefrom 40-80° C., preferably 60-80° C., and preferably at pH 4-5, saidvariant comprising one or more mutation(s) in the following position(s)or region(s) in the amino acid sequence shown in NO: 2:

[0299] Region: 1-18,

[0300] Region: 19-35,

[0301] Region: 73-80,

[0302] Region: 93-127,

[0303] Region: 170-184,

[0304] Region: 200-212,

[0305] Region: 234-246,

[0306] Region: 287-319,

[0307] Region: 334-341,

[0308] Region: 353-374,

[0309] Region: 388-414,

[0310] Region: 445-470,

[0311] and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: N20C, A27C, S30P, A246C.

[0312] As substrate binding may improve the stability region 93-127,Region: 170-184, Region: 305-319 are also contemplated forthermostabilization according to the present invention.

[0313] In an embodiment the region mutated is the Region: 1-18.

[0314] Specific preferred positions contemplated include one or more of:

[0315] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.

[0316] Specific mutations include one or more of: A1V, T2P/Q/R/H/M/E,N9A, A11E/P.

[0317] Preferred combinations of mutations include one or more of:

[0318] A1V+L66R+Y402F+N427S+S486G,

[0319] T2K+S30P+N427M+S44G+V470M,

[0320] T2E+T379A+S386K+A393R,

[0321] T2R+S386R+A393R,

[0322] A11P+T2Q+S394R,

[0323] T2Q+A11P+S394R,

[0324] T2R+L66V+S394P+Y402F,

[0325] T2M+N9A+T390R+D406N+L410R,

[0326] T2R+S386R+A393R,

[0327] A11E+E408R.

[0328] In an embodiment the region mutated is the Region: 19-35.

[0329] Preferred sub-regions include one or more of: 21-26, 31-35.

[0330] Specific preferred positions contemplated include one or more of:19, 21, 22, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35.

[0331] Specific mutations include one or more of: L19N, N20T, G23A,A24S/T, D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V, D33R/K/H,S34N.

[0332] In an embodiment the region mutated is the Region: 73-80.

[0333] Specific preferred positions contemplated include one or more of:

[0334] 73, 74, 75, 76, 77, 78, 79, 80.

[0335] Specific mutations include one or more of:

[0336] S73P/D/T/N/Q/E,

[0337] L74I/D,

[0338] L75A/R/N/D/C/Q/E/G/H/I/K/M/P/S/T/V, preferred are I/N/D,

[0339] S76A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred T/A)

[0340] T77V/T,

[0341] I78V,

[0342] E79A/R/N/D/C/Q/G/H/I/L/K/F/M/P/S/T/Y/V, preferred are Q/R/K)

[0343] N80A/R/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred are

[0344] H/D/E/R/K/T/S/Y.

[0345] In an embodiment the region mutated is the Region: 93-127.

[0346] In an additional embodiment the sub-region is: Region: 93-124

[0347] Preferred sub-regions include one or more of: 93-107, 109-111,113-115.

[0348] Specific preferred positions contemplated include one or more of:

[0349] 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,109, 110, 111, 113, 114, 115, 117, 118, 126, 127.

[0350] Preferred mutations include one or more of:

[0351] P107M/L/A/G/S/T/V/I,

[0352] S119A/R/N/D/Q/H/I/L/K/F/M/T/V, preferred is A,

[0353] R122A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

[0354] Preferred combinations of mutations include one or more of:S119P+G447S. S119P+A393R.

[0355] In an embodiment the region mutated is the Region: 170-184.

[0356] Specific mutations include one or more of:

[0357] N171R/K/Q/E/W/F/Y,

[0358] Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

[0359] T173K/R/S/N/Q,

[0360] G174A, S,

[0361] Y175N/Q/D/E,

[0362] D176L

[0363] L177I

[0364] E180N/M

[0365] V181I/T

[0366] N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V.

[0367] G183A

[0368] S184D/N/E/Q/L/I/T/R/K.

[0369] In an embodiment the region mutated is the Region: 200-212.

[0370] Preferred sub-regions include: 200-211.

[0371] Specific preferred positions contemplated include one or more of:

[0372] 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

[0373] Specific mutations include one or more of: A203L, S211P.

[0374] In an embodiment the region mutated is the Region: 234-246.

[0375] Preferred sub-regions include one or more of: 234-240, 242-245.

[0376] Specific preferred positions contemplated include one or more of:

[0377] 234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

[0378] Specific mutations include one or more of:

[0379] L234A/R/N/D/C/Q/E/G/H/I/K/M/F/P/S/T/W/Y/V,

[0380] A235S,

[0381] F237Y/H/N/D,

[0382] D238T/S,

[0383] S239A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V,

[0384] S240G,

[0385] S242S/P/T/A/Y/H/N/D,

[0386] G243S/P/T/A/Y/H/N/D,

[0387] K244R,

[0388] A246T.

[0389] Preferred combinations of mutations include one or more of:A246T+T721.

[0390] In an embodiment the region mutated is the Region: 287-319.

[0391] Preferred sub-regions include one or more of: 287-292, 294-301,313-316.

[0392] In an additional embodiment the sub-region include: 305-319

[0393] Specific preferred positions contemplated include one or more of:

[0394] 287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

[0395] Specific mutations include one or more of:

[0396] Y312Q,

[0397] F318A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/T/Y/V, preferred is Y.

[0398] Preferred combinations of mutations include one or more of:

[0399] N313G+F318Y, Y302Q+S119P+T416H+Y402F.

[0400] In an embodiment the region mutated is the Region: 334-341.

[0401] Specific preferred positions contemplated include one or more of:

[0402] 334, 335, 336, 337, 338, 339, 340, 341.

[0403] Specific mutations include one or more of:

[0404] D336A/R/N/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V.

[0405] K337AR/N/D/C/Q/E/G/H/I/L/M/F/P/S/T/W/Y/V.

[0406] Q338A/R/N/D/C/G/H/I/L/F/P/S/T/Y/V.

[0407] G339S/P/T/A.

[0408] S340I/T/N/I/A/D/G.

[0409] L341F/L/I/V.

[0410] Preferred combinations of mutations include one or more of:

[0411] S340G+D357S+T360V+S386P.

[0412] In an embodiment the region mutated is the Region: 353-374.

[0413] Specific preferred positions contemplated include one or more of:

[0414] 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374.

[0415] Specific mutations include one or more of:

[0416] A353D/S,

[0417] S356P/N/D,

[0418] D357S,

[0419] A359S,

[0420] T360V,

[0421] G361S/P/T/A,

[0422] T362R,

[0423] S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0424] S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0425] S366T,

[0426] S368P/T/A,

[0427] T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,

[0428] S371Y/H/N/D,

[0429] S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G.

[0430] Preferred combinations of mutations include one or more of:

[0431] S356P+S366T,

[0432] D357S+T360V+S371H

[0433] D357S+T360V+S386P+S340G.

[0434] In an embodiment the region mutated is the Region: 388-414.

[0435] Preferred sub-regions include one or more of: 397-399, 402-406,412-414.

[0436] In an additional embodiment the sub-region is: 407-411

[0437] Specific preferred positions contemplated include one or more of:

[0438] 388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409,412, 413, 414.

[0439] Specific mutations include one or more of: T390R,

[0440] A393R,

[0441] S394P/R,

[0442] S399A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/T/W/Y/V, preferred are T/Q/C,

[0443] Y402A/R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/V, preferred is F,

[0444] D403S,

[0445] S405T,

[0446] D406N,

[0447] E408C/R,

[0448] Q409A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V, preferred is P,

[0449] L410I/R,

[0450] S411V,

[0451] A412C,

[0452] R413A/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V.

[0453] D414A.

[0454] Preferred combinations of mutations include one or more of:

[0455] A393R+T2R+S386R,

[0456] A393R+T490A+V59A+PLASD(N-terminal extension),

[0457] S394R+T2Q+A11P,

[0458] Y402F+T2R+L66V+S394P,

[0459] Y402F+S411V+S119P

[0460] Y402F+S411V,

[0461] Y402F+312Q+S119P+T416H,

[0462] S411V+A393R,

[0463] E408R+S386N,

[0464] E408R+A425T+S465P+T494A,

[0465] L410R+A393R,

[0466] S411V+S119P+402F+A393R,

[0467] S411V+S119P+402F+A393R+T416H.

[0468] In an embodiment the region mutated is the Region: 445-470.

[0469] Preferred sub-regions include one or more of: 445-459, 461-470.

[0470] Specific preferred positions contemplated include one or more of:

[0471] 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 461, 462, 463, 464, 465, 466, 467, 467, 468, 469, 470.

[0472] Specific mutations include one or more of: G447S, G456C/P, S465P.

[0473] Preferred combinations of mutations include one or more of:

[0474] G447S+S119P,

[0475] S465P+E408R+A425T+T494A.

[0476] Increased Specific Activity

[0477] In a third aspect the invention relates to a variant of a parentglucoamylase with increased specific activity comprising one or moremutation(s) in the following position(s) or region(s) in the amino acidsequence shown in NO: 2:

[0478] Region: 1-18,

[0479] Region: 40-62,

[0480] Region: 93-127,

[0481] Region: 170-184,

[0482] Region: 200-212,

[0483] Region: 234-246,

[0484] Region: 287-319,

[0485] Region: 388-414,

[0486] and/or in a corresponding position or region in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2, with the exception of the followingsubstitutions: S411G.

[0487] In an embodiment the region mutated is the Region: 1-18.

[0488] Specific preferred positions contemplated include one or more of:

[0489] 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19.

[0490] Specific mutations are: L3N, 118V.

[0491] Preferred combinations of mutations include one or more of:

[0492] I18V+T51S+S56A+V59T+L60A.

[0493] In an embodiment the region mutated is the Region: 40-62.

[0494] Preferred sub-regions include one or more of: 40-47, 58-62.

[0495] Specific preferred positions contemplated include one or more of:40, 41, 42, 43, 44, 45, 46, 47, 49, 51, 53, 56, 58, 59, 60, 61, 62.

[0496] Specific mutations include one or more of:

[0497] S40C, T43R, T51S/D, T53D, S56A/C, V59T, L60A.

[0498] Preferred combinations of mutations include one or more of:

[0499] T51S+S56A+V59T+L60A+I18V.

[0500] In an embodiment the region mutated is the Region: 93-127.

[0501] In an additional embodiment the sub-region is: Region: 93-124

[0502] Preferred sub-regions include one or more of: 93-107, 109-111,113-115.

[0503] Specific preferred positions contemplated include one or more of:

[0504] 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,109, 110, 111, 113, 114, 115, 117, 118, 126, 127.

[0505] Specific mutations include one or more of: N93T, P94V, S95N,D97S, L98S/P, S100T/D, A102S/*, N110T, V111P, D112N,E113M/A, T114S,A115Q/G, Y116F, S119A, G127A.

[0506] Preferred combinations of mutations include one or more of:

[0507] S119P+Y402F,

[0508] S119P+Y402F+I189T+Y223F+F227Y.

[0509] In an embodiment the region mutated is the Region: 170-184.

[0510] Specific mutations include one or more of:

[0511] N171R/K/Q/E/W/F/Y,

[0512] Q172A/R/N/D/C/E/G/H/I/L/K/F/M/P/S/T/W/Y/V,

[0513] T173K/R/S/N/Q,

[0514] G174A, S,

[0515] Y175N/Q/D/E,

[0516] D176L

[0517] L177I

[0518] E180N/M

[0519] V181I/T

[0520] N182R/C/E/G/H/I/L/K/M/P/T/W/Y/V, preferred is E,

[0521] G183A

[0522] S184D/N/E/Q/L/I/M/T/R/K.

[0523] In an embodiment the region mutated is the Region: 200-212.

[0524] Preferred sub-regions include: 200-211.

[0525] Specific preferred positions contemplated include one or more of:

[0526] 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211.

[0527] Specific mutations include one or more of: A201D, F202L, A203L,T204K, A205R/S, V206L/N, G207N, S208H/T/D, S209T, W212N/A/T.

[0528] In an embodiment the region mutated is the Region: 234-246.

[0529] Preferred sub-regions include one or more of: 234-240, 242-245.

[0530] Specific preferred positions contemplated include one or more of:

[0531] 234, 235, 236, 237, 238, 239, 240, 242, 243, 244, 245.

[0532] In an embodiment the region mutated is the Region: 287-319.

[0533] Preferred sub-regions include one or more of: 287-292, 294-301,313-316.

[0534] Specific preferred positions contemplated include one or more of:

[0535] 287, 288, 289, 290, 291, 292, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319.

[0536] Specific mutations include one or more of:

[0537] S287A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

[0538] I288L/N/Q,

[0539] Y289F,

[0540] T290A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/V,

[0541] L291I/D/N,

[0542] N292D,

[0543] D293A/R/N/C/Q/E/G/H/I/L/K/M/S/T/V,

[0544] G294A/R/N/D/C/Q/E/H/I/L/K/M/P/S/T/V,

[0545] L295A/R/N/D/C/Q/E/G/H/K/M/S/T/V,

[0546] S296A/R/N/D/C/Q/E/G/H/I/L/K/M/T/V,

[0547] D297A/R/N/C/Q/E/G/H/I/L/K/M/P/S/T/V,

[0548] S298A/R/N/D/C/Q/E/G/H/I/L/K/F/M/T/V,

[0549] E299A/R/N/D/C/Q/G/H/I/L/K/M/S/T/V.

[0550] V301T/I,

[0551] A302R/N/D/C/Q/E/G/H/I/L/K/F/M/P/S/T/W/Y/V/, preferred S, V303T/I,

[0552] G304A,

[0553] R305A/N/D/C/Q/E/G/H/I/L/F/M/P/S/T/W/Y/V,

[0554] Y306A/R/N/D/C/Q/E/G/H/I/L/K/M/P/S/T/W/Y/V.

[0555] E308A/R/N/D/C/Q/G/H/I/L/K/M/P/S/T/V, preferred Q,

[0556] D309L,

[0557] T310V/S,

[0558] Y311N,

[0559] Y312Q/N, preferred is Q,

[0560] N313T/S/G, preferred is S,

[0561] N315Q/E/R.

[0562] In an embodiment the region mutated is the Region: 388-414.

[0563] Preferred sub-regions include one or more of: 397-399, 402-406,412-414.

[0564] Specific preferred positions contemplated include one or more of:

[0565] 388, 389, 390, 394, 397, 398, 399, 402, 403, 404, 405, 406, 409,412, 413, 414.

[0566] Specific mutations include one or more of: M398L, S399C/Q/T,Y402F, D403S, S405T, E408C/R, S411V, A412C, D414A.

[0567] Preferred combinations of mutations include one or more of:

[0568] Y402F+S119P,

[0569] Y402F+S119P+I189T+Y223F+F227Y.

[0570] In a preferred embodiment of the invention the regions to bemutated are:

[0571] Region: 287-300,

[0572] Region: 305-319,

[0573] and/or corresponding positions or regions in a homologousglucoamylase which displays at least 60% homology with the amino acidsequences shown in SEQ ID NO: 2.

[0574] Homology (Identity)

[0575] The homology referred to above of the parent glucoamylase isdetermined as the degree of identity between two protein sequencesindicating a derivation of the first sequence from the second. Thehomology may suitably be determined by means of computer programs knownin the art such as GAP provided in the GCG program package (ProgramManual for the Wisconsin Package, Version 8, August 1994, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p.443-453). Using Gap with the following settings for polypeptide sequencecomparison: Gap creation penalty of 3.0 and Gap extension penalty of0.1, the mature part of a polypeptide encoded by an analogous DNAsequence of the invention exhibits a degree of identity preferably of atleast 60%, such as 70%, at least 80%, at least 90%, more preferably atleast 95%, more preferably at least 97%, and most preferably at least99% with the mature part of the amino acid sequence shown in SEQ ID NO:2.

[0576] Preferably, the parent glucoamylase comprise the amino acidsequences of SEQ ID NO: 2; or allelic variants thereof; or fragmentsthereof that has glucoamylase activity.

[0577] A fragment of SEQ ID NO: 2 is a polypeptide which have one ormore amino acids deleted from the amino and/or carboxyl terminus of thisamino acid sequence. For instance, the AMG G2 (SEQ ID NO: 2) is afragment of the Aspergillus niger G1 glucoamylase (Boel et al. (1984),EMBO J. 3 (5), p. 1097-1102) having glucoamylase activity. An allelicvariant denotes any of two or more alternative forms of a gene occupyingthe same chromosomal locus. Allelic variation arises naturally throughmutation, and may result in polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or mayencode polypeptides having altered amino acid sequences. An allelicvariant of a polypeptide is a polypeptide encoded by an allelic variantof a gene.

[0578] The amino acid sequences of homologous parent glucoamylases maydiffer from the amino acid sequence of SEQ ID NO: 2 by an insertion ordeletion of one or more amino acid residues and/or the substitution ofone or more amino acid residues by different amino acid residues.Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

[0579] In another embodiment, the isolated parent glucoamylase isencoded by a nucleic acid sequence which hybridises under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with a nucleic acidprobe which hybridises under the same conditions with (i) the nucleicacid sequence of SEQ ID NO: 1, (ii) the cDNA sequence of SEQ ID NO: 1,(iii) a sub-sequence of (i) or (ii), or (iv) a complementary strand of(i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y). The sub-sequence of SEQ ID NO: 1 may be at least 100 nucleotidesor preferably at least 200 nucleotides. Moreover, the sub-sequence mayencode a polypeptide fragment which has glucoamylase activity. Theparent polypeptides may also be allelic variants or fragments of thepolypeptides that have glucoamylase activity.

[0580] The nucleic acid sequence of SEQ ID NO: 1 or a subsequencethereof, as well as the amino acid sequence of SEQ ID NO: 2, or afragment thereof, may be used to design a nucleic acid probe to identifyand clone DNA encoding polypeptides having glucoamylase activity, fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

[0581] Thus, a genomic DNA or cDNA library prepared from such otherorganisms may be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having glucoamylase.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilised on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, or sub-sequences thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridisation indicates that the nucleic acid sequencehybridises to a nucleic acid probe corresponding to the nucleic acidsequence shown in SEQ ID NO: 1 its complementary strand, or asub-sequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridises under theseconditions are detected using X-ray film.

[0582] For long probes of at least 100 nucleotides in length, thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS preferably at least at 45° C. (very low stringency),more preferably at least at 50° C. (low stringency), more preferably atleast at 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

[0583] For short probes which are about 15 nucleotides to about 70nucleotides in length, stringency conditions are defined asprehybridization, hybridisation, and washing post-hybridization at 50°C. to 10° C. below the calculated T_(m) using the calculation accordingto Bolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

[0584] For short probes which are about 15 nucleotides to about 70nucleotides in length, the carrier material is washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 50°C. to 10° C. below the calculated T_(m).

[0585] The present invention also relates to isolated nucleic acidsequences produced by (a) hybridising a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with the sequenceof SEQ ID NO: 1, or its complementary strand, or a sub-sequence thereof;and (b) isolating the nucleic acid sequence. The sub-sequence ispreferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has glucoamylase activity.

[0586] Contemplated parent glucoamylases have at least 20%, preferablyat least 40%, more preferably at least 60%, even more preferably atleast 80%, even more preferably at least 90%, and most preferably atleast 100% of the glucoamylase activity of the mature polypeptide of SEQID NO: 2.

[0587] In a preferred embodiment the variant of the invention hasimproved thermal stability and/or increased specific activity,preferably within the temperature interval from about 60-80° C.,preferably 63-75° C., preferably at a pH of 4-5, in particular 4.2-4.7,using maltodextrin as the substrate.

[0588] In another preferred embodiment a variant of the invention isused for, e.g., alcohol fermentation.

[0589] In a preferred embodiment the parent glucoamylase is theAspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.1097-1102. The parent glucoamylase may be a truncated glucoamylase,e.g., the AMG G2 glucoamylase.

[0590] Cloning a DNA Sequence Encoding A Parent Glucoamylase

[0591] The DNA sequence encoding a parent glucoamylase may be isolatedfrom any cell or microorganism producing the glucoamylase in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the glucoamylase to be studied.Then, if the amino acid sequence of the glucoamylase is known, labeledoligonucleotide probes may be synthesized and used to identifyglucoamylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to another known glucoamylase gene couldbe used as a probe to identify glucoamylase-encoding clones, usinghybridization and washing conditions of very low to very highstringency. This is described above.

[0592] Yet another method for identifying glucoamylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming glucoamylase-negative bacteriawith the resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for glucoamylase (i.e.,maltose), thereby allowing clones expressing the glucoamylase to beidentified.

[0593] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g. thephosphoroamidite method described S. L. Beaucage and M. H. Caruthers,(1981), Tetrahedron Letters 22, p. 1859-1869, or the method described byMatthes et al., (1984), EMBO J. 3, p. 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors. Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al., (1988), Science 239, 1988, pp. 487-491.

[0594] Site-directed Mutagenesis

[0595] Once a glucoamylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites. In a specificmethod, a single-stranded gap of DNA, the glucoamylase-encodingsequence, is created in a vector carrying the glucoamylase gene.

[0596] Then the synthetic nucleotide, bearing the desired mutation, isannealed to a homologous portion of the single-stranded DNA. Theremaining gap is then filled in with DNA polymerase I (Klenow fragment)and the construct is ligated using T4 ligase. A specific example of thismethod is described in Morinaga et al., (1984), Biotechnology 2, p.646-639. U.S. Pat. No. 4,760,025 discloses the introduction ofoligonucleotides encoding multiple mutations by performing minoralterations of the cassette. However, an even greater variety ofmutations can be introduced at any one time by the Morinaga method,because a multitude of oligonucleotides, of various lengths, can beintroduced.

[0597] Another method for introducing mutations intoglucoamylase-encoding DNA sequences is described in Nelson and Long,(1989), Analytical Biochemistry 180, p. 147-151. It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

[0598] Further, Sierks. et al., (1989), Protein Eng., 2, 621-625; Sierkset al., (1990), Protein Eng. vol. 3, 193-198; also describessite-directed mutagenesis in an Aspergillus glucoamylase.

[0599] Random Mutagenesis

[0600] Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

[0601] The random mutagenesis of a DNA sequence encoding a parentglucoamylase may be conveniently performed by use of any method known inthe art.

[0602] In relation to the above, a further aspect of the presentinvention relates to a method for generating a variant of a parentglucoamylase, wherein the variant exhibits increased thermal stabilityrelative to the parent, the method comprising:

[0603] (a) subjecting a DNA sequence encoding the parent glucoamylase torandom mutagenesis,

[0604] (b) expressing the mutated DNA sequence obtained in step (a) in ahost cell, and

[0605] (c) screening for host cells expressing a glucoamylase variantwhich has an altered property (i.e. thermal stability) relative to theparent glucoamylase.

[0606] Step (a) of the above method of the invention is preferablyperformed using doped primers, as described in the working examplesherein (vide infra).

[0607] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

[0608] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) ir-radiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues. When such agents areused, the mutagenesis is typically performed by incubating the DNAsequence encoding the parent enzyme to be mutagenized in the presence ofthe mutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

[0609] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding theglucoamylase enzyme by any published technique, using, e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

[0610] Preferably, the doping is carried out using “constant randomdoping”, in which the percentage of wild-type and mutation in eachposition is predefined. Furthermore, the doping may be directed toward apreference for the introduction of certain nucleotides, and thereby apreference for the introduction of one or more specific amino acidresidues. The doping may be made, e.g., so as to allow for theintroduction of 90% wild type and 10% mutations in each position. Anadditional consideration in the choice of a doping scheme is based ongenetic as well as protein-structural constraints. The doping scheme maybe made by using the DOPE program which, inter alia, ensures thatintroduction of stop codons is avoided.

[0611] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent glucoamylase is subjectedto PCR under conditions that increase the mis-incorporation ofnucleotides (Deshler 1992; Leung et al., Technique, Vol. 1, 1989, pp.11-15).

[0612] A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding theglucoamylase by, e.g., transforming a plasmid containing the parentglycosylase into the mutator strain, growing the mutator strain with theplasmid and isolating the mutated plasmid from the mutator strain. Themu-ta-ted plasmid may be subsequently transformed into the expressionorganism.

[0613] The DNA sequence to be mutagenized may be conveniently present ina genomic or cDNA library prepared from an organism expressing theparent glucoamylase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or other-wise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0614] In some cases it may be convenient to amplify the mutated DNAsequence prior to performing the expression step b) or the screeningstep c). Such amplification may be performed in accordance with methodsknown in the art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

[0615] Subsequent to the incubation with or exposure to the mutagenisingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which carried the DNA sequence encoding the parent enzymeduring the mutagenesis treatment. Examples of suitable host cells arethe following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

[0616] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

[0617] Localized Random Mutagenesis

[0618] The random mutagenesis may be advantageously localized to a partof the parent glucoamylase in question. This may, e.g., be advantageouswhen certain regions of the enzyme have been identified to be ofparticular importance for a given property of the enzyme, and whenmodified are expected to result in a variant having improved properties.Such regions may normally be identified when the tertiary structure ofthe parent enzyme has been elucidated and related to the function of theenzyme.

[0619] The localized, or region-specific, random mutagenesis isconveniently performed by use of PCR generated mutagenesis techniques asdescribed above or any other suitable technique known in the art.Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by insertion into a suitable vector,and said part may be subsequently subjected to mutagenesis by use of anyof the mutagenesis methods discussed above.

[0620] Alternative methods for providing variants of the inventioninclude gene shuffling e.g. as described in WO 95/22625 (from AffymaxTechnologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S).

[0621] Expression Of Glucoamylase Variants

[0622] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

[0623] Expression Vector

[0624] The recombinant expression vector carrying the DNA sequenceencoding a glucoamylase variant of the invention may be any vector whichmay conveniently be subjected to recombinant DNA procedures, and thechoice of vector will often depend on the host cell into which it is tobe introduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated. Examples ofsuitable expression vectors include pMT838.

[0625] Promoter

[0626] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

[0627] Examples of suitable promoters for directing the transcription ofthe DNA sequence encoding a glucoamylase variant of the invention,especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis _(α)-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens _(α)-amylase(amyQ), the promoters of the Bacillus subtilis xyIA and xyIB genes etc.For transcription in a fungal host, examples of useful promoters arethose derived from the gene encoding A. oryzae TAKA amylase, the TPI(triose phosphate isomerase) promoter from S. cerevisiae (Alber et al.(1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei asparticproteinase, A. niger neutral _(α)-amylase, A. niger acid stable_(α)-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzaealkaline protease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase.

[0628] Expression Vector

[0629] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding the_(α)-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[0630] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB 110, pE194, pAMB1 and pIJ702.

[0631] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g. as described in WO 91/17243.

[0632] The procedures used to ligate the DNA construct of the inventionencoding a glucoamylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

[0633] Host Cells

[0634] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of aglucoamylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0635] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

[0636] Examples of suitable bacteria are Gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

[0637] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.

[0638] The host cell may also be a filamentous fungus e.g. a strainbelonging to a species of Aspergillus, most preferably Aspergillusoryzae or Aspergillus niger, or a strain of Fusarium, such as a strainof Fusarium oxysporium, Fusarium graminearum (in the perfect state namedGribberella zeae, previously Sphaeria zeae, synonym with Gibberellaroseum and Gibberella roseum f. sp. cerealis), or Fusarium sulphureum(in the prefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellnse), or Fusarium venenatum.

[0639] In a preferred embodiment of the invention the host cell is aprotease deficient or protease minus strain.

[0640] This may for instance be the protease deficient strainAspergillus oryzae JaL 125 having the alkaline protease gene named “alp”deleted. This strain is described in WO 97/35956 (Novo Nordisk).

[0641] Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host micro-organism is described in EP 238 023 (NovoNordisk A/S), the contents of which are hereby incorporated byreference.

[0642] Method of Producing a Glucoamylase Variant

[0643] In a yet further aspect, the present invention relates to amethod of producing a glucoamylase variant of the invention, whichmethod comprises cultivating a host cell under conditions conducive tothe production of the variant and recovering the variant from the cellsand/or culture medium.

[0644] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the glucoamylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g. as described in catalogues of the American TypeCulture Collection).

[0645] The glucoamylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

[0646] Starch Conversion

[0647] The present invention provides a method of using glucoamylasevariants of the invention for producing glucose and the like fromstarch. Generally, the method includes the steps of partiallyhydrolyzing precursor starch in the presence of _(α)-amylase and thenfurther hydrolyzing the release of D-glucose from the non-reducing endsof the starch or related oligo- and polysaccharide molecules in thepresence of glucoamylase by cleaving _(α)-(1

4) and _(α)-(1

6) glucosidic bonds.

[0648] The partial hydrolysis of the precursor starch utilizinga-amylase provides an initial breakdown of the starch molecules byhydrolyzing internal _(α)-(1

4)-linkages. In commercial applications, the initial hydrolysis using_(α)-amylase is run at a temperature of approximately 105° C. A veryhigh starch concentration is processed, usually 30% to 40% solids. Theinitial hydrolysis is usually carried out for five minutes at thiselevated temperature. The partially hydrolyzed starch can then betransferred to a second tank and incubated for approximately 1-2 hour ata temperature of 85° to 98° C. to derive a dextrose equivalent (D.E.) of10 to 15.

[0649] The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30° and 62° C. Preferablythe temperature of the substrate liquid is dropped to between 55° and60° C. The pH of the solution is dropped from about 5.5 to 6.5 to arange between 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5.The glucoamylase is added to the solution and the reaction is carriedout for 24-72 hours, preferably 36-48 hours.

[0650] By using a thermostable glucoamylase variant of the inventionsaccharification processes may be carried out at a higher temperaturethan traditional batch saccharification processes. According to theinvention saccharification may be carried out at temperatures in therange from above 60-80° C., preferably 63-75° C. This apply both fortraditional batch processes (described above) and for continuoussaccharification processes.

[0651] Actually, continuous saccharification processes including one ormore membrane separation steps, i.e. filtration steps, must be carriedout at temperatures of above 60° C. to be able to maintain a reasonablyhigh flux over the membrane or to minimize microbial contamination.Therefore, the thermostable variants of the invention provides thepossibility of carrying out large scale continuous saccharificationprocesses at a fair price and/or at a lower enzyme protein dosage withina period of time acceptable for industrial saccharification processes.According to the invention the saccharification time may even beshortened.

[0652] The activity of the glucoamylase variant (e.g. AMG variant) ofthe invention is generally substantially higher at temperatures between60° C.-80° C. than at the traditionally used temperature between 30-60°C. Therefore, by increasing the temperature at which the glucoamylaseoperates the saccharification process may be carried out within ashorter period of time.

[0653] Further, by improving the thermal stability the T_(½) (half-time,as defined in the “Materials and Methods” section) is improved. As thethermal stability of the glucoamylase variants of the invention isimproved a minor amount of glucoamylase need to be added to replace theglucoamylase being inactivated during the saccharification process. Moreglucoamylase is maintained active during saccharification processaccording to the present invention. Furthermore, the risk of microbialcontamination is also reduced when carrying the saccharification processat temperature above 63° C.

[0654] The glucose yield from a typical saccharification trial withglucoamylase, acid amylase and pullulanase is 95.5-96.5%. The remainingcarbohydrates typically consists of 1% maltose, 1.5-2% isomaltose and1-1.5% higher oligosacharides. The disaccharides are produced since theglucoamylase at high concentrations of glucose and high dry-solid levelshas a tendency to form reversion products.

[0655] A glucoamylase with an increased specific activity towardssaccharides present in the solution after liquefaction and saccharidesformed during saccharification would be an advantage as a reduced enzymeprotein dosage or a shorter process time then could be used. In general,the glucoamylase has a preference for substrates consisting of longersaccharides compared to short chain saccharides and the specificactivity towards e.g. maltoheptaose is therefore approximately 6 timeshigher than towards maltose. An increased specific activity towardsshort chain saccharides such as maltose (without reducing the activitytowards oligosaccharides) would therefore also permit using a lowerenzyme dosage and/or shorter process time.

[0656] Furthermore, a higher glucose yield can be obtained with aglucoamylase variant with an increased alpha-1,4 hydrolytic activity (ifthe alpha-1,6 activity is unchanged or even decreased), since a reducedamount of enzyme protein is being used, and alpha-1,6 reversion productformation therefore is decreased (less isomaltose).

[0657] The specific activity may be measured using the method describedin the “Materials & Methods” section at 37° C. or 60° C.

[0658] Example of saccharification process wherein the glucoamylasevariants of the invention may be used include the processes described inJP 3-224493; JP 1-191693; JP 62-272987; EP 452, 238, and WO 99/27124(all references are hereby incorporated by reference).

[0659] In a further aspect the invention relates to a method ofsaccharifying a liquefied starch solution, comprising the steps

[0660] (i) a saccharification step during which step one or moreenzymatic saccharification stages takes place, and the subsequent stepof

[0661] (ii) one or more high temperature membrane separation steps

[0662] herein the enzymatic saccharification is carried out using athermostable lucoamylase variant of the invention.

[0663] The glucoamylase variant(s) of the invention may be used in thepresent inventive process in combination with an enzyme that hydrolyzesonly _(α)-(1

6)-glucosidic bonds in molecules with at least four glucosyl residues.Preferentially, the glucoamylase variant of the invention can be used incombination with pullulanase or isoamylase. The use of isoamylase andpullulanase for debranching, the molecular properties of the enzymes,and the potential use of the enzymes with glucoamylase is set forth inG. M. A. van Beynum et al., Starch Conversion Technology, Marcel Dekker,New York, 1985, 101-142.

[0664] In a further aspect the invention relates to the use of aglucoamylase variant of the invention in a starch conversion process.

[0665] Further, the glucoamylase variant of the invention may be used ina continuous starch conversion process including a continuoussaccharification step.

[0666] The glucoamylase variants of the invention may also be used inimmobilised form. This is suitable and often used for producingspeciality syrups, such as maltose syrups, and further for the raffinatestream of oligosaccharides in connection with the production of fructosesyrups.

[0667] The glucoamylase of the invention may also be used in a processfor producing ethanol for fuel or beverage or may be used in afermentation process for producing organic compounds, such as citricacid, ascorbic acid, lysine, glutamic acid.

MATERIALS & METHODS MATERIALS

[0668] Enzymes:

[0669] AMG G1: Aspergillus niger glucoamylase G1 disclosed in Boel etal. (1984), EMBO J. 3 (5), 1097-1102, and SEQ ID NO: 13, available fromNovo Nordisk.

[0670] AMG G2: Truncated Aspergillus niger glucoamylase G1 shown in SEQID NO: 2, available from Novo Nordisk)

[0671] Solutions:

[0672] Buffer: 0.05M sodium acetate (6.8 g in 1 l milli-Q-water), pH 4.5

[0673] Stop solution: 0.4M NaOH

[0674] GOD-perid, 124036, Boehringer Mannheim

[0675] Substrate:

[0676] Maltose: 29 mM (1 g maltose in 100 ml 50 mM sodium acetate, pH4.5) (Sigma)

[0677] Maltoheptaose: 10 mM, 115 mg/10 ml (Sigma)

[0678] Host cell:

[0679]A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available fromInstitute for Fermention, Osaka; 17-25 Juso Hammachi 2-ChomeYodogawa-ku, Osaka, Japan, having the alkaline protease gene named “alp”(described by Murakami K et al., (1991), Agric. Biol. Chem. 55, p.2807-2811) deleted by a one step gene replacement method (described byG. May in “Applied Molecular Genetics of Filamentous Fungi” (1992), p.1-25. Eds. J. R. Kinghorn and G. Turner; Blackie Academic andProfessional), using the A. oryzae pyrG gene as marker. Strain JaL 125is further disclosed in WO 97/35956 (Novo Nordisk).

[0680] Micro-organisms:

[0681] Strain: Saccharomyces cerevisiae YNG318: MAT_(α)leu2-_(Δ)2ura3-52 his4-539 pep4-_(Δ)1[cir+]

[0682] Plasmids:

[0683] pCAMG91: see FIG. 1. Plasmid comprising the Aspergillus niger G1glucoamylase (AMG G1). The construction of pCAMG91 is described in Boelet al. (1984), EMBO J. 3 (7) p. 1581-1585.

[0684] pMT838: Plasmid encoding the truncated Aspergillus nigerglucoamylase G2 (SEQ ID NO: 2).

[0685] pJSO026 (S. cerevisiae expression plasmid)(J. S. Okkels, (1996)“AURA3-promoter deletion in a pYES vector increases the expression levelof a fungal lipase in Saccharomyces cerevisiae. Recombinant DNABiotechnology III: The Integration of Biological and EngineeringSciences, vol. 782 of the Annals of the New York Academy of Sciences)More specifically, the expression plasmid pJSO37, is derived from pYES2.0 by replacing the inducible GAL1-promoter of pYES 2.0 with theconstitutively expressed TPI (triose phosphate isomerase)-promoter fromSaccharomyces cerevisiae (Albert and Karwasaki, (1982), J. Mol. ApplGenet., 1, 419-434), and deleting a part of the URA3 promoter.

Method

[0686] Transformation of Saccharomyces cerevisiae YNG318

[0687] The DNA fragments and the opened vectors are mixed andtransformed into the yeast Saccharomyces cerevisiae YNG318 by standardmethods.

[0688] Determining Specific Activity As k_(cat) (sec. ⁻¹).

[0689] 750 microL substrate (1% maltose, 50 mM Sodium acetat, pH 4.3) isincubated 5 minutes at selected temperature, such as 37° C. or 60° C.

[0690] 50 microL enzyme diluted in sodium acetate is added. Aliquots of100 microL are removed after 0, 3, 6, 9 and 12 minutes and transferredto 100 microL 0.4 M Sodium hydroxide to stop the reaction. A blank isincluded. 20 microL is transferred to a Micro titre plates and 200microL GOD-Perid solution is added. Absorbance is measured at 650 nmafter 30 minutes incubation at room temperature.

[0691] Glucose is used as standard and the specific activity iscalculated as k_(cat) (sec. ⁻¹).

[0692] Determination of AGU Activity and as AGU/mg

[0693] One Novo Amyloglucosidase Unit (AGU) is defined as the amount ofenzyme which hydrolyzes 1 micromole maltose per minute at 37° C. and pH4.3. A detailed description of the analytical method (AEL-SM-0131) isavailable on request from Novo Nordisk.

[0694] The activity is determined as AGU/ml by a method modified after(AEL-SM-0131) using the Glucose GOD-Perid kit from Boehringer Mannheim,124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml.

[0695] 375 microL substrate (1% maltose in 50 mM Sodium acetate, pH 4.3)is incubated 5 minutes at 37° C. 25 microL enzyme diluted in sodiumacetate is added. The reaction is stopped after 10 minutes by adding 100microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitreplate and 200 microL GOD-Perid solution is added. After 30 minutes atroom temperature, the absorbance is measured at 650 nm and the activitycalculated in AGU/ml from the AMG-standard.

[0696] The specific activity in AGU/mg is then calculated from theactivity (AGU/ml) divided with the protein concentration (mg/ml).

[0697] Transformation of Aspergillus oryzae (General Procedure)

[0698] 100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics,Cold Spring Harbor Laboratory) are inoculated with spores of A. oryzaeand incubated with shaking for about 24 hours. The mycelium is harvestedby filtration through miracloth and washed with 200 ml of 0.6 M MgSO₄.The mycelium is suspended in 15 ml of 1.2 M MgSO₄, 10 mM NaH₂PO₄, pH5.8. The suspension is cooled on ice and 1 ml of buffer containing 120mg of Novozym™ 234 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigmatype H25) is added and incubation with gentle agitation continued for1.5-2.5 hours at 37C until a large number of protoplasts is visible in asample inspected under the microscope.

[0699] The suspension is filtered through miracloth, the filtratetransferred to a sterile tube and overlayed with 5 ml of 0.6 M sorbitol,100 mM Tris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000g and the protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally, the protoplasts areresuspended in 0.2-1 ml of STC.

[0700] 100 μl of protoplast suspension are mixed with 5-25 μg of p3SR2(an A. nidulans amdS gene carrying plasmid described in Hynes et al.,Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, August 1983) in 10 μl ofSTC. The mixture is left at room temperature for 25 min. 0.2 ml of 60%PEG 4000 (BDH 29576), 10 mM CaCl₂ and 10 mM Tris-HCl, pH 7.5 is addedand carefully mixed (twice) and finally 0.85 ml of the same solution areadded and carefully mixed. The mixture is left at room temperature for25 min., spun at 2.500 g for 15 min. and the pellet is resuspended in 2ml of 1.2M sorbitol. After one more sedimentation the protoplasts arespread on minimal plates (Cove, (1966), Biochem. Biophys. Acta 113,51-56) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogensource and 20 mM CsCl to inhibit background growth. After incubation for4-7 days at 37° C. spores are picked, suspended in sterile water andspread for single colonies. This procedure is repeated and spores of asingle colony after the second re-isolation are stored as a definedtransformant.

[0701] Fed Batch Fermentation

[0702] Fed batch fermentation is performed in a medium comprisingmaltodextrin as a carbon source, urea as a nitrogen source and yeastextract. The fed batch fermentation is performed by inoculating a shakeflask culture of A. oryzae host cells in question into a mediumcomprising 3.5% of the carbon source and 0.5% of the nitrogen source.After 24 hours of cultivation at pH 5.0 and 34° C. the continuous supplyof additional carbon and nitrogen sources are initiated. The carbonsource is kept as the limiting factor and it is secured that oxygen ispresent in excess. The fed batch cultivation is continued for 4 days,after which the enzymes can be recovered by centrifugation,ultrafiltration, clear filtration and germ filtration.

[0703] Purification

[0704] The culture broth is filtrated and added ammoniumsulphate (AMS)to a concentration of 1.7 M AMS and pH is adjusted to pH 5. Precipitatedmaterial is removed by centrifugation and the solution containingglucoamylase activity is applied on a Toyo Pearl Butyl column previouslyequilibrated in 1.7 M AMS, 20 mM sodium acetate, pH 5. Unbound materialis washed out with the equilibration buffer. Bound proteins are elutedwith 10 mM sodium acetate, pH 4.5 using a linear gradient from 1.7-0 MAMS over 10 column volumes. Glucoamylase containing fractions arecollected and dialysed against 20 mM sodium acetate, pH 4.5. Thesolution was then applied on a Q sepharose column, previouslyequilibrated in 10 mM Piperazin, Sigma, pH 5.5. Unbound material iswashed out with the equilibration buffer. Bound proteins are eluted witha linear gradient of 0-0.3 M Sodium chloride in 10 mM Piperazin, pH 5.5over 10 column volumes. Glucoamylase containing fractions are collectedand the purity was confirmed by SDS-PAGE.

[0705] T_(½) (half-life) Method I

[0706] The thermal stability of variants is determined as T_(½) usingthe following method: 950 microliter 50 mM sodium acetate buffer (pH4.3) (NaOAc) is incubated for 5 minutes at 68° C. or 70° C. 50microliter enzyme in buffer (4 AGU/ml) is added. 2×40 microliter samplesare taken at 0, 5, 10, 20, 30 and 40 minutes and chilled on ice. Theactivity (AGU/ml) measured before incubation (0 minutes) is used asreference (100%). The decline in stability (in percent) is calculated asa function of the incubation time. The % residual glucoamylase activityis determined at different times. T_(½) is the period of time untilwhich the % relative activity is decreased to 50%.

[0707] T_(½) (half-life) (Method II)

[0708] The T_(½) is measured by incubating the enzyme (ca 0.2 AGU/ml) inquestion in 30% glucose, 50 mM Sodium acetate at pH 4.5 at thetemperature in question (e.g., 70° C.). Samples are withdrawn at settime intervals and chilled on ice and residual enzyme activity measuredby the pNPG method (as described below).

[0709] The % residual glucoamylase activity is determined at differenttimes. T_(½) is the period of time until which the % relative activityis decreased to 50%.

[0710] Residual Enzyme Activity (pNPG Method)

[0711] pNPG reagent:

[0712] 0.2 g pNPG (p-nitrophenylglucopyranoside) is dissolved in 0.1 Macetate buffer (pH 4.3) and made up to 100 ml.

[0713] Borate solution:

[0714] 3.8 g Na₂B₄O₇10 H₂O is dissolved in Milli-Q water and made up to100 ml.

[0715] 25 microL samples are added 50 microL substrate and incubated 2hr at 50° C. The reaction is stopped by adding 150 micoL ml boratesolution. The optical density is at 405 nm, and the residual activitycalculated.

[0716] Construction of pAMGY

[0717] The pAMGY vector was constructed as follows: The lipase gene inpJSO026 was replaced by the AMG gene, which was PCR amplified with theforward primer; FG2: 5′-CAT CCC CAG GAT CCT TAC TCA GCA ATG-3′ and thereverse primer: RG2: 5′-CTC AAA CGA CTC ACC AGC CTC TAG AGT-3′ using thetemplate plasmid pLAC103 containing the AMG gene. The pJSO026 plasmidwas digested with Xbal and Smal at 37° C. for 2 hours and the PCRamplicon was blunt ended using the Klenow fragment and then digestedwith Xbal. The vector fragment and the PCR amplicon were ligated andtransformed into E. coli by electrotransformation. The resulting vectoris designated pAMGY.

[0718] Construction of pLaC103

[0719] The A. niger AMGII cDNA clone (Boel et al., (1984), supra) isused as source for the construction of pLaC103 aimed at S. cerevisiaeexpression of the GII form of AMG.

[0720] The construction takes place in several steps, out lined below.

[0721] pT7-212 (EP37856/U.S. Pat. No. 5,162,498) is cleaved with Xbal,blunt-ended with Klenow DNA polymerase and dNTP. After cleavage withEcoRI the resulting vector fragment is purified from an agarosegel-electrophoresis and ligated with the 2.05 kb EcoR1-EcoRV fragment ofpBoel53, thereby recreating the Xbal site in the EcoRV end of the AMGencoding fragment in the resulting plasmid pG2x.

[0722] In order to remove DNA upstream of the AMG cds, and furnish theAMG encoding DNA with an appropriate restriction endonucleaserecognition site, the following construct was made:

[0723] The 930 bp EcoRI-PstI fragment of p53 was isolated and subjectedto Alul cleavage, the resulting 771 bp Alu-PstI fragment was ligatedinto pBR322 with blunt-ended EcoRI site (see above) and cleaved withPstI In the resulting plasmid pBR-AMG′, the EcoRI site was recreatedjust 34 bp from the initiation codon of the AMG cds.

[0724] From pBR-AMG′ the 775 bp EcoRI-PstI fragment was isolated andjoined with the 1151 bp PstI-XbaI fragment from pG2x in a ligationreaction including the XbaI-EcoRI vector fragment of pT7-212.

[0725] The resulting plasmid pT7GII was submitted to a BamHI cleavage inpresence of alkaline phosphatase followed by partial SphI cleavage afterinactivation of the phosphatase. From this reaction was the 2489 bpSphI-BamHI fragment, encompassing the S.c. TPI promoter linked to theAMGII cds.

[0726] The above fragment together with the 1052 bp BamHI fragment ofpT7GII was ligated with the alkaline phosphatase treated vector fragmentof pMT743 (EP37856/U.S. Pat. No. 5,162,498), resulting from SphI-BamHIdigestion. The resulting plasmid is pLaC103.

[0727] Screening for Thermostable AMG Variants

[0728] The libraries are screened in the thermostable filter assaydescribed below.

[0729] Filter Assay for Thermostability

[0730] Yeast libraries are plated on a sandwich of cellulose acetate (OE67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on SCFura agarplates with 100 μg/ml ampicillin at 30° C. for at least 72 hrs. Thecolonies are replica plated to PVDF filters (Immobilon-P, Millipore,Bedford) activated with methanol for 1 min or alternatively a Protranfilter (no activation) and subsequently washed in 0.1 M NaAc and thenincubated at room temperature for 2 hours. Colonies are washed fromPVDF/Protran filters with tap water. Each filter sandwiches andPVDF/Protran filters are specifically marked with a needle beforeincubation in order to be able to localise positive variants on thefilters after the screening. The PVDF filters with bound variants aretransferred to a container with 0.1 M NaAc, pH 4.5 and incubated at 47°C. or alternatively 67-69° C. in case of Protran filters for 15 minutes.The sandwich of cellulose acetate and nitrocellulose filters on SCura-agar plates are stored at room temperature until use. Afterincubation, the residual activities are detected on plates containing 5%maltose, 1% agarose, 50 mM NaAc, pH 4.5. The assay plates with PVDFfilters are marked the same way as the filter sandwiches and incubatedfor 2 hrs. at 5° C. After removal of the PVDF filters, the assay platesare stained with Glucose GOD perid (Boehringer Mannheim GmbH, Germany).Variants with residual activity are detected on assay plates as darkgreen spots on white background. The improved variants are located onthe storage plates. Improved variants are rescreened twice under thesame conditions as the first screen.

[0731] General Method for Random Mutagenesis by Use of the DOPE Program

[0732] The random mutagenesis may be carried out by the following steps:

[0733] 1. Select regions of interest for modification in the parentenzyme,

[0734] 2. Decide on mutation sites and non-mutated sites in the selectedregion,

[0735] 3. Decide on which kind of mutations should be carried out, e.g.,with respect to the desired stability and/or performance of the variantto be constructed,

[0736] 4. Select structurally reasonable mutations,

[0737] 5. Adjust the residues selected by step 3 with regard to step 4.

[0738] 6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

[0739] 7. If necessary, adjust the wanted residues to genetic coderealism, e.g. taking into account constraints resulting from the geneticcode, e.g. in order to avoid introduction of stop codons; the skilledperson will be aware that some codon combinations cannot be used inpractice and will need to be adapted

[0740] 8. Make primers

[0741] 9. Perform random mutagenesis by use of the primers

[0742] 10. Select resulting glucoamylase variants by screening for thedesired improved properties.

[0743] Dope Algorithm

[0744] Suitable dope algorithms for use in step 6 are well known in theart. One such algorithm is described by Tomandl, D. et al., 1997,Journal of Computer-Aided Molecular Design 11:29-38. Another algorithmis DOPE (Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T(1998) Nucleic Acids Research 26:697-702).

[0745] Method Of Extracting Important Regions For Temperature ActivityUsing Molecular Simulation.

[0746] The X-ray structure and/or the model-build structure of theenzyme of interest, here AMG, are subjected to molecular dynamicssimulations. The molecular dynamics simulation are made using the CHARMM(from Molecular simulations (MSI)) program or other suitable programs,e.g., DISCOVER (from MSI). The dynamics are made in vacuum, or includingcrystal waters, or with the enzyme in question embedded in a suitablewaters, e.g., a sphere or a box. The simulation are run for 300picoseconds (ps) or more, e.g., 300-1200 ps. The isotropic fluctuationsare extracted for the CA carbons of the structures and comparisonbetween the structures are made. More details on how to get theisotropic fluctuations can be found in the CHARMM manual (available fromMSI) and hereby incorporated herein by reference.

[0747] The molecular dynamics simulation can be carried out usingstandard charges on the chargeable amino acids. For instance, Asp andGlu is negatively charged and Lys and Arg are positively charged. Thiscondition resembles the medium pH of approximately 7.0. To analyze alower pH, titration of the molecule can be done to obtain the alteredpKa's of the normal titrateable residues within pH 2-10; Lys, Arg, Asp,Glu, Tyr and His. Also Ser, Thr and Cys are titrateable but are nottaking into account here. Here the altered charges due to the pH hasbeen described as all Arg, Lys negative at high pH, and all Asp, Glu areuncharged. This imitates a pH around 4 to 5 where the titration Asp andGlu normally takes place. Model building of the enzyme of interest canbe obtained by using the HOMOLOGY model in the MSI program package. Thecrystal structure of Aspergillus awamori variant X100 can be found in,e.g., 3GLY and 1DOG in the Brookhaven database.

EXAMPLES Example 1

[0748] Construction of AMG G2 Variants

[0749] Site-directed mutagenesis:

[0750] For the construction of variants of AMG G2 (SEQ ID NO: 2) thecommercial kit, Chameleon double-stranded, site-directed mutagenesis kitwas used according to the manufacturer's instructions.

[0751] The gene encoding the AMG G2 enzyme in question is located onpMT838 prepared by deleting the DNA between G2 nt. 1362 and G2 nt. 1530in plasmid pCAMG91 (see FIG. 1) comprising the AMG G1 form.

[0752] In accordance with the manufacturer's instructions the Scal siteof the Ampicillin gene of pMT838 was changed to a Mlul site by use ofthe following primer: 7258: 5′p gaa tga ctt ggt tga cgc gtc acc agt cac3′ (SEQ ID NO: 3). (Thus changing the Scal site found in the ampicillinresistance gene and used for cutting to a Mlul site). The pMT838 vectorcomprising the AMG gene in question was then used as a template for DNApolymerase and oligo 7258 (SEQ ID NO: 3) and 21401 (SEQ ID NO: 4).

[0753] Primer no. 21401 (SEQ ID NO: 4) was used as the selection primer.21401: 5′p gg gga tca tga tag gac tag cca tat taa tga agg gca tat accacg cct tgg acc tgc gtt ata gcc 3′

[0754] (Changes the Scal site found in the AMG gene without changing theamino acid sequence).

[0755] The desired mutation (e.g., the introduction of a cysteinresidue) is introduced into the AMG gene in question by addition ofappropriate oligos comprising the desired mutation.

[0756] The primer 107581 was used to introduce T12P 107581: 5′ pgc aacgaa gcg ccc gtg gct cgt ac 3′ (SEQ ID NO: 5)

[0757] The mutations are verified by sequencing the whole gene. Theplasmid was transformed into A. oryzae using the method described abovein the “Materials & Methods” section. The variant was fermented andpurified as described above in the “Materials & Methods” section.

Example 2

[0758] Construction, by Localized random, Doped Mutagenesis, of A nigerAMG Variants Having Improved Thermostability Compared to the ParentEnzyme

[0759] To improve the thermostability of the A. niger AMG randommutagenesis in pre-selected region was performed.

[0760] Residue:

[0761] Region: L19-G35

[0762] Region: A353-V374

[0763] The DOPE software (see Materials and Methods) was used todetermine spiked codons for each suggested change in the above regionsminimizing the amount of stop codons (see table 1). The exactdistribution of nucleotides was calculated in the three positions of thecodon to give the suggested population of amino acid changes. The dopedregions were doped specifically in the indicated positions to have ahigh chance is of getting the desired residues, but still allow otherpossibilities.

[0764] The first column is the amino acid to be mutated, the secondcolumn is the percentage of wild type and the third column defined thenew amino acid(s). TABLE 1 Doping in L19-G35 L19   90% N N20   95% T N21Constant I22 Constant G23   95% A A24   90% S, T D25   93% S, T, R G26  95% A A27   90% S, T W28 <80% R, Y V29 Constant S30   93% T, N G31  95% A A32   95% V D33   80% R, K, H S34   90% N G35 Constant

[0765] The resulting doped oligonucleotide strand is shown in table 2 assense strand: with the primer sequence, the wild type nucleotidesequence, the parent amino acid sequence and the distribution ofnucleotides for each doped position. TABLE 2 Position:  19   20    2122      23    24  25  26  27 A.a.seq.:  L    N     N I   G     A D G   Aprimer: 12T   A3T   AAC   ATC   G4G   5CG 67C G4T 8CT wt.seq.:CTG   AAT   AAC   ATC   GGG   GCG GAC GGT GCT Pos.(cont.):28         2930      31    32    33      34    35 A.a.(cont.):W  V     S  G      A         D    S    G primer: 91010   GTG  1112CG4C  G13G 141516 1718T    GGC Wt seq.:TGG     GTG  TCG  GGC   GCG   GAC     TCT   GGC

[0766] Distribution of nucleotides for each doped position.

[0767] 1: A10, C90

[0768] 2: A6, T94

[0769] 3: A95, C5

[0770] 4: G95, C5

[0771] 5: G91, A3, T3, C3

[0772] 6: G95, A3, C2

[0773] 7: G3, A95, C2

[0774] 8: G92, A4, T4

[0775] 9: A3, T97

[0776] 10: G95, T5

[0777] 11: G3, A97

[0778] 12: G95, A2, C3

[0779] 13: T5, C95

[0780] 14: G88, A8, C4

[0781] 15: G7, A93

[0782] 16: G4, C96

[0783] 17: G4, A96

[0784] 18: G95, A2, C3

[0785] Forward Primer (SEQ ID NO: 6):

[0786] FAMGII ′5-C GAA GCG ACC GTG GCT CGT ACT GCC ATC 12T A3T AAC ATCG4G 5CG 67C G4T 8CT 91010 GTG 1112C G4C G13G 141516 1718T GGC ATT GTCGTT GCT AGT CCC AGC ACG GAT AAC-3′

[0787] Reverse primer (SEQ ID NO: 7):

[0788] RAMG1: 5′-GAT GGC AGT ACG AGC CAC GGT CGC TTC G-3′ TABLE 3 Dopingin region A353-V374: A353 <80% D, E, Q, N, Y L354   90% Q, E Y355   90%N, Q S356   90% T, D, N G357   80% P, A, S, T A358   93% S A359   90% S,T, N T360   90% R, K G361   85% A, S, T T362   90% S Y363 Constant S364  93% D S365   93% N, Q, K S366   93% P, D S367 Constant S368   93% D,N, T T369   93% Q, E Y370 Constant S371   93% N S372   93% N, T I373Constant V374   93% N, Y, H

[0789] The resulting doped oligonucleotide strand is shown in table 4 assense strand: with the primer sequence, wild type nucleotide sequence,the parent amino acid sequence and the distribution of nucleotides foreach doped position. TABLE 4 Position: 353     354 355 356357    358    359     360   361   362 A.a.seq.:A L Y S     D  A  A   T  G       T primer: 123 45A 6AC 78C 910T 11CT1213T 1415A   1617C 18CC Wt. seq.: GCA CTG TAC AGC GAT  GCT GCT ACTGGC          ACC Pos. (cont.): 363    364   365    366 367368369     370 A.a. seq.(cont.): Y     S   S  S    S S          T  Y primer(cont.): TAC 1920T A2122 2324C AGT 1425C 2627G T28T wt. Seq.(cont.): TACTCT  TCG  TCC  AGT TCG     ACT  TAT Pos. (cont.): 371     372     373374 A.a.pos. (cont.): S     S   I V primer (cont.) A16T 2930T ATT 313233wt. Seq.(cont.): AGT AGC ATT GTA

[0790] Distribution of nucleotides for each doped position.

[0791] 1: G91, A3, T3, C3

[0792] 2: A13, C87

[0793] 3: A40, T60

[0794] 4: G3, A3, C94

[0795] 5: A6, T94

[0796] 6: G4, A4, T92

[0797] 7: G2, A96, C2

[0798] 8: G93, A3.5, C3.5

[0799] 9: G87, A8, C5

[0800] 10: A84, C16

[0801] 11: G93, T7

[0802] 12: G92, A5, T3

[0803] 13: A3, C97

[0804] 14: G3, A97

[0805] 15: G2, A2, T4, C92

[0806] 16: G93, A7

[0807] 17: G93, C7

[0808] 18: A90, T10

[0809] 19: G4, A96

[0810] 20: G95, A5

[0811] 21: G96, A4

[0812] 22: G3, C97

[0813] 23: G2, A1, T95, C2

[0814] 24: A3, C97

[0815] 25: G95, A3, C2

[0816] 26: G2, A96, C2

[0817] 27: A5, C95

[0818] 28: A95, T5

[0819] 29: G2, A98

[0820] 30: G94, A4, C2

[0821] 31: G94, A3, T1, C2

[0822] 32: A4, T96

[0823] 33: A20, C80

[0824] Primer: FAMGIV (SEQ ID NO: 8)

[0825] 5′-GTG TCG CTG GAC TTC TTC AAG 123 45A 6AC 78C 910T 11CT 1213T1415A 1617C 18CC TAC 1920T A2122 2324C AGT 1425C 2627G T28T A16T 2930CATT 313233 GAT GCC GTG AAG ACT TTC GCC GA-3′

[0826] Primer RAMGVI (SEQ ID NO: 9)

[0827] 5′-ctt gaa gaa gtc cag cga cac-3′

[0828] Random Mutagenesis

[0829] The spiked oligonucleotides apparent from Table 2 and 3 (which bya common term is designated FAMG) and reverse primers RAMG for theL19-G35 region and specific SEQ ID NO: 2 primers covering the N-terminal(FG2: 5′-CAT CCC CAG GAT CCT TAC TCA GCA ATG-3′ (SEQ ID NO: 10) andC-terminal (RG2: 5′-CTC AAA CGA CTC ACC AGC CTC TAG AGT (SEQ ID NO: 11)are used to generate PCR-library-fragments by the overlap extensionmethod (Horton et al., Gene, 77 (1989), pp. 61-68) with an overlap of 21base pairs. Plasmid pAMGY is template for the Polymerase Chain Reaction.The PCR fragments are cloned by homologous recombination in the E.coli/yeast shuttle vector pAMGY (see Materials and Methods).

[0830] Screening

[0831] The library was screened in the thermostability filter assaysusing a Protran filter and incubating at 67-69° C. as described in the“Material & Methods” section above

Example 3

[0832] Construction, by PCR Shuffling Spiked with DNA Oligos, of A.niger AMG Variants Having Improved Thermostability Compared to theParent Enzyme

[0833] The polymerase chain reaction (PCR) method was used to prepareDNA fragments carrying the AMG gene and flanking regions. Approximately10 ug DNA was digested with Dnase, and run on a 2% agarose gel.Fragments of 50-150 bp were purified from the gel. Approximately 1 ugpurified fragments were mixed with a 5-15 fold molar excess of oligoscarrying the desired mutations. The oligos were of the following kind(for the construction of Hklib1, Hklib2, Hklib3 etc., respectively):

[0834] Hklib1:

[0835] Hk1-T2X:

[0836] 5′-ATGTGATTTCCAAGCGCGCGVNNTTGGATTCATGGTTGAGCAA (SEQ ID NO: 14)

[0837] Hk1-N9X:

[0838] 5′-CCTTGGATTCATGGTTGAGCVNNGAAGCGACCGTGGCTCGTAC (SEQ ID NO: 15)

[0839] Hk1-A11X:

[0840] 5′-ATTCATGGTTGAGCAACGAAVNNACCGTGGCTCGTACTGCCAT (SEQ ID NO: 16)

[0841] Hk1-L66X:

[0842] 5′-TCCTCAAGACCCTCGTCGATVNNTTCCGAAATGGAGATACCAG (SEQ ID NO: 17)

[0843] Hk1-S386X:

[0844] 5′-CTTTCGCCGATGGCTTCGTCVNNATTGTGGAAACTCACGCCGC (SEQ ID NO: 18)

[0845] Hk1-E389X:

[0846] 5′-ATGGCTTCGTCTCTATTGTGVNNACTCACGCCGCAAGCAACGG (SEQ ID NO: 19)

[0847] Hk1-T390X:

[0848] 5′-GCTTCGTCTCTATTGTGGAAVNNCACGCCGCAAGCAACGGCTC (SEQ ID NO: 20)

[0849] Hk1-A393X:

[0850] 5′-CTATTGTGGAAACTCACGCCVNNAGCAACGGCTCCATGTCCGA (SEQ ID NO: 21)

[0851] Hk1-S394X:

[0852] 5′-TTGTGGAAACTCACGCCGCAVNNAACGGCTCCATGTCCGAGCA (SEQ ID NO: 22)

[0853] Hk1-N395X:

[0854] 5′-TGGAAACTCACGCCGCAAGCVNNGGCTCCATGTCCGAGCAATA (SEQ ID NO: 23)

[0855] Hk1-G396X:

[0856] 5′-AAACTCACGCCGCAAGCAACVNNTCCATGTCCGAGCAATACGA (SEQ ID NO: 24)

[0857] Hk1-K404X:

[0858] 5′-CCATGTCCGAGCAATACGACVNNTCTGATGGCGAGCAGCTTTC (SEQ ID NO: 25)

[0859] Hk1-D406X:

[0860] 5′-CCGAGCAATACGACAAGTCTVNNGGCGAGCAGCTTTCCGCTCG (SEQ ID NO: 26)

[0861] Hk1-E408X:

[0862] 5′-AATACGACAAGTCTGATGGCVNNCAGCTTTCCGCTCGCGACCT (SEQ ID NO: 27)

[0863] Hk1-L410X:

[0864] 5′-ACAAGTCTGATGGCGAGCAGVNNTCCGCTCGCGACCTGACCT (SEQ ID NO: 28)

[0865] Hk1-L423X:

[0866] 5′-CCTGGTCTTATGCTGCTCTGVNNACCGCCAACAACCGTCGTAA (SEQ ID NO: 29)

[0867] Hk1-N426X:

[0868] 5′-ATGCTGCTCTGCTGACCGCCVNNAACCGTCGTAACTCCGTCGTG (SEQ ID NO: 30)

[0869] Hk1-N427X:

[0870] 5′-CTGCTCTGCTGACCGCCAACVNNCGTCGTAACTCCGTCGTGCCT (SEQ ID NO: 31)

[0871] Hk1-Y402X:

[0872] 5′-ACGGCTCCATGTCCGAGCAANNCGACAAGTCTGATGGCGAGCAGCT (SEQ ID NO: 32)

[0873] Hklib2:

[0874] Hk2-L234X-SENSE:

[0875] 5′-CTGGACCGGCAGCTTCATTNNKGCCAACTTCGATAGCAGCC (SEQ ID NO: 33)

[0876] Hk2-A235S-ANTISENSE:

[0877] 5′-GAACGGCTGCTATCGAAGTTAGACAGAATGAAGCTGCCGGTC (SEQ ID NO: 34)

[0878] Hk2-F237X-SENSE: 5-CAGCTTCATTCTGGCCAACNATGATAGCAGCCGTTCCGGCA (SEQID NO: 35)

[0879] Hk2-D238T-ANTISENSE:

[0880] 5′-CCTTGCCGGAACGGCTGCTAGTGAAGTTGGCCAGAATGAAGC (SEQ ID NO: 36)

[0881] Hk2-D238S-ANTISENSE:

[0882] 5′-CCTTGCCGGAACGGCTGCTAGAGAAGTTGGCCAGAATGAAGC (SEQ ID NO: 37)

[0883] Hk2-S239X-SENSE:

[0884] 5′-TCATTCTGGCCAACTTCGATNNCAGCCGTTCCGGCAAGGACG (SEQ ID NO: 38)

[0885] Hk2-S240G-ANTISENSE:

[0886] 5′-TTGCGTCCTTGCCGGAACGACCGCTATCGAAGTTGGCCAGAA (SEQ ID NO: 39)

[0887] Hk2-S242X-ANTISENSE:

[0888] 5′-GGGTGTTTGCGTCCTTGCCAKNACGGCTGCTATCGAAGTTG (SEQ ID NO: 40)

[0889] Hk2-G243X-ANTISENSE:

[0890] 5′-GGAGGGTGTTTGCGTCCTTAKNGGAACGGCTGCTATCGAAG (SEQ ID NO: 41)

[0891] Hk2-K244R-SENSE:

[0892] 5′-CGATAGCAGCCGTTCCGGCAGAGACGCAAACACCCTCCTGG (SEQ ID NO: 42)

[0893] Hk2-T310V-ANTISENSE:

[0894] 5′-ACGGGTTGCCGTTGTAGTAAACGTCCTCAGGGTACCGACCC (SEQ ID NO: 43)

[0895] Hk2-T310S-ANTISENSE:

[0896] 5′-ACGGGTTGCCGTTGTAGTAAGAGTCCTCAGGGTACCGACCC (SEQ ID NO: 44)

[0897] Hk2-Y311N-SENSE:

[0898] 5′-TCGGTACCCTGAGGACACGAATTACAACGGCAACCCGTGGT (SEQ ID NO: 45)

[0899] Hk2-Y312Q-ANTISENSE:

[0900] 5′-GGAACCACGGGTTGCCGTTTTGGTACGTGTCCTCAGGGTAC (SEQ ID NO: 46)

[0901] Hk2-Y312N-ANTISENSE:

[0902] 5′-GGAACCACGGGTTGCCGTTATTGTACGTGTCCTCAGGGTAC (SEQ ID NO: 47)

[0903] Hk2-N313T-SENSE:

[0904] 5′-CCCTGAGGACACGTACTACACTGGCAACCCGTGGTTCCTGT (SEQ ID NO: 48)

[0905] Hk2-N313S-SENSE:

[0906] 5′-CCCTGAGGACACGTACTACTCTGGCAACCCGTGGTTCCTGT (SEQ ID NO: 49)

[0907] Hk2-N313G-SENSE:

[0908] 5′-CCCTGAGGACACGTACTACGGTGGCAACCCGTGGTTCCTGT (SEQ ID NO: 50)

[0909] Hk2-N315Q-ANTISENSE:

[0910] 5′-AGGTGCACAGGGAACCACGGTTGGCCGTTGTAGTACGTGTCC (SEQ ID NO: 51)

[0911] Hk2-N315E-ANTISENSE:

[0912] 5′-AGGTGCACAGGAACCACGGTTCGCCGTTGTAGTACGTGTCC (SEQ ID NO: 52)

[0913] Hk2-N315R-ANTISENSE:

[0914] 5′-AGGTGCACAGGAACCACGGTCTGCCGTTGTAGTACGTGTCC (SEQ ID NO: 53)

[0915] Hk2-F318Y-ANTISENSE:

[0916] 5′-CGGCAGCCAAGGTGCACAGATACCACGGGTTGCCGTTGTAG (SEQ ID NO: 54)

[0917] Hk2-Q409P-SENSE:

[0918] 5′-CGACAAGTCTGATGGCGAGCCACTTTCCGCTCGCGACCTGA (SEQ ID NO: 55)

[0919] Hklib3:

[0920] Hk3-D336X-SENSE:

[0921] 5′-CGATGCTCTATACCAGTGGNNKKAAGCAGGGGTCGTTGGAGG (SEQ ID NO: 56)

[0922] Hk3-K337X-SENSE:

[0923] 5′-TGCTCTATACCAGTGGGACNNKCAGGGGTCGTTGGAGGTCA (SEQ ID NO: 57)

[0924] Hk3-Q338X-ANTISENSE:

[0925] 5′-CTGTGACCTCCAACGACCCGNNCTTGTCCCACTGGTATAGA (SEQ ID NO: 58)

[0926] Hk3-G339X-SENSE:

[0927] 5′-ATACCAGTGGGACAAGCAGNCUTCGTTGGAGGTCACAGATG (SEQ ID NO: 59)

[0928] Hk3-S340X′-ANTISENSE:

[0929] 5′-ACACATCTGTGACCTCCAAANTCCCCTGCTTGTCCCACTGG (SEQ ID NO: 60)

[0930] Hk3-S340X″-ANTISENSE:

[0931] 5′-ACACATCTGTGACCTCCAAANCCCCCTGCTTGTCCCACTGG (SEQ ID NO: 61)

[0932] Hk3-L341X-SENSE:

[0933] 5′-GTGGGACAAGCAGGGGTCGNUUGAGGTCACAGATGTGTCGC (SEQ ID NO: 62)

[0934] Hk3-K352Q-SENSE:

[0935] 5′-TGTGTCGCTGGACTTCTTCCAAGCACTGTACAGCGATGCTG (SEQ ID NO: 63)

[0936] Hk3-K352R-SENSE:

[0937] 5′-TGTGTCGCTGGACTTCTTCAGAGCACTGTACAGCGATGCTG (SEQ ID NO: 64)

[0938] Hk3-A353D-ANTISENSE:

[0939] 5′-TAGCAGCATCGCTGTACAGATCCTTGAAGAAGTCCAGCGAC (SEQ ID NO: 65)

[0940] Hk3-A353S-ANTISENSE:

[0941] 5′-TAGCAGCATCGCTGTACAGAGACTTGAAGAAGTCCAGCGAC (SEQ ID NO: 66)

[0942] Hk3-S356P-SENSE:

[0943] 5′-ACTTCTTCAAGGCACTGTACCCAGATGCTGCTACTGGCACCT (SEQ ID NO: 67)

[0944] Hk3-S356N-SENSE:

[0945] 5′-ACTTCTTCAAGGCACTGTACAAUGATGCTGCTACTGGCACCTA (SEQ ID NO: 68)

[0946] Hk3-S356D-SENSE:

[0947] 5′-ACTTCTTCAAGGCACTGTACGAUGATGCTGCTACTGGCACCTA (SEQ ID NO: 69)

[0948] Hk3-D357S-ANTISENSE:

[0949] 5′-GAGTAGGTGCCAGTAGCAGCAGAGCTGTACAGTGCCTTGAAGA (SEQ ID NO: 70)

[0950] Hk3-A359S-SENSE:

[0951] 5′-GGCACTGTACAGCGATGCTTCTACTGGCACCTACTCTTCGT (SEQ ID NO: 71)

[0952] Hk3-T360V-ANTISENSE:

[0953] 5′-TGGACGAAGAGTAGGTGCCAACAGCAGCATCGCTGTACAGT (SEQ ID NO: 72)

[0954] Hk3-G361X-SENSE:

[0955] 5′-TGTACAGCGATGCTGCTACTNCTACCTACTCTTCGTCCAGTTC (SEQ ID NO: 73)

[0956] Hk3-T362R-ANTISENSE:

[0957] 5′-GTCGAACTGGACGAAGAGTATCTGCCAGTAGCAGCATCGCTG (SEQ ID NO: 74)

[0958] Hk3-S364X-SENSE:

[0959] 5′-TGCTGCTACTGGCACCTACNNKTCGTCCAGTTCGACTTATAG (SEQ ID NO: 75)

[0960] Hk3-S365X-SENSE:

[0961] 5′-TGCTACTGGCACCTACTCTNNKTCCAGTTCGACTTATAGTAG (SEQ ID NO: 76)

[0962] Hk3-S366T-ANTISENSE:

[0963] 5′-ATGCTACTATAAGTCGAACTAGTCGAAGAGTAGGTGCCAGTA (SEQ ID NO: 77)

[0964] Hk3-S368X-ANTISENSE:

[0965] 5′-TCTACAATGCTACTATAAGTAGNACTGGACGAAGAGTAGGTG (SEQ ID NO: 78)

[0966] Hk3-T369X-SENSE:

[0967] 5′-CTACTCTTCGTCCAGTTCGNNKTATAGTAGCATTGTAGATGCC (SEQ ID NO: 79)

[0968] Hk3-S371X-ANTISENSE:

[0969] 5′-TTCACGGCATCTACAATGCTATNATAAGTCGAACTGGACGAAG (SEQ ID NO: 80)

[0970] Hk3-S372X-SENSE:

[0971] 5′-CGTCCAGTTCGACTTATAGTNNTATTGTAGATGCCGTGAAGAC (SEQ ID NO: 81)

[0972] To the mix of Dnase treated DNA and oligos was added nucleotides,PCR buffer and Taq/Pwo polymerase. A PCR assembly reaction wasperformed, using first 94° C. for 2 min., then 35-40 cycles with thefollowing incubation times: 94° C., 30 sec.; 45° C., 30 sec.; 72° C., 60sec; then finally 72° C. for 5 min.

[0973] An PCR amplification reaction was performed with 1 uL of theassembly reaction as template, and adding primers that anneal to theregions flanking the AMG gene. Parameters: first 94° C. for 2 min., then35-40 cycles with the following incubation times: 94° C., 30 sec.; 55°C., 30 sec.; 72° C., 90 sec; then finally 72° C. for 10 min.

[0974] The resulting PCR product was purified from a 1% agarose gel,mixed with linearized vector and transformed into competent yeast cells,as described above.

Example 4

[0975] Specific Activity

[0976] AMG G2 variants were constructed as described above in Example 1.The specific activity as k_(cat) were measured on purified samples at pH4.3, 37° C., using maltose and maltohepatose as substrate as describedin the “Materials & Methods” section above. The specific activity asAGU/mg were also measured at pH 4.3, 37° C., using maltose as substrateas described in the “Materials & Methods” section above. Kcat (sec. −1)Variant Maltose Maltoheptaose AMG G2 (wt) 6.0 38 N110T 9.7 27.8 V111P12.0 43.2 S119P 6.2 44.0 G127A 21.0 40.0 G207N 30.5 36.3

[0977] Variant AGU/mg AMG G2 (wild type) 1.8 N110T 3.5 V111P 3.1 S119P2.1 G127A 5.8 G207N 5.7 L3N 2.3 S56A 2.6 A102* 2.5 D403S 2.2 I18V +T51S + S56A + V59T + L60A 3.3 S119P + Y402F 2.7 S119P + I189T + Y223F +F227Y + Y402F 3.0

Example 5

[0978] Thermostability at 70° C.

[0979] An AMG G2 S119P variant was constructed using the approachdescribed in Example 1.

[0980] The thermostability was determined as T_(½) using Method I, andas % residual activity after incubation for 30 minutes in 50 mM NaOAc,pH 4.5, 70° C., 0.2 AGU/ml, as described in the “Material & Methods”section above. The result of the tests are listed in the Table below andcompared to the wild-type A. niger AMG G2. Residual T½ A. niger AMG(Enzyme) activity (%) (min.) S119P variant 22 17 wild-type (SEQ ID NO:2) 13  8

Example 6

[0981] Thermostability at 68° C.

[0982] AMG G2 variants were constructed using the approach described inExample 3, except for variants nos. 1 and 2 in the Table below, whichwere prepared by shuffling as described in WO 95/22625 (from AffymaxTechnologies N.V.).

[0983] The thermostability was determined as T½ using method I at 68° C.as described in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants were performed on culture broth after filtration of thesupernatants. T½ A. niger AMG G2 T½ (wild type) Variant (min) (min)  1A246T + T72I 11.3 8.5  2 G447S + S119P 11.4 7.9  3 E408R + A425T +S465P + T494A 8.6 8.1  4 E408R + S386N 12.6 8.9  5 T2P 9.3 8.5  6 T2Q +A11P + S394R 10.7 8.5  7 T2H 9.5 8.9  8 A11E + E408R 12.7 9.3  9 T2M +N9A + T390R + D406N + L410R 10.7 8.5 10 A393R 17.7 8.4 11 T2R + S386R +A393R 14.1 8.4 12 A393R + L410R 14.7 7.9 13 A1V + L66R + Y402F + N427S +S486G 11.7 8.5 14 T2K + S30P + N427M + S444G + V470M 11.4 8.4

[0984] Thermostability at 70° C. on purified samples. Enzyme T½ (min) 15AMG G2 (wild type) 7.4  16 T2E + T379A + S386K + A393R 11.6 17 E408R +S386N 10.2 18 T2Q + A11P + S394R 9.8  19 A1V + L66R + Y402F + N427S +S486G 14.1 20 A393R 14.6 21 T2R + S386R + A393R 14.1 22 A393R + L410R12.9 23 Y402F 10.1

Example 7

[0985] Thermostability at 68° C.

[0986] AMG G2 variants were constructed by shuffling using the approachdescribed in Example 3 followed by shuffling of positive variants.

[0987] The thermostability was determined as T½ using method I at 68° C.as described in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants were performed on culture broth after filtration of thesupernatants. T½ A. niger AMG G2 T½ (wild type) Variant (min) (min) 24PLASD^(i) + V59A + A393R + T490A 27.2 6.8

Example 8

[0988] Thermostability at 68° C.

[0989] AMG G2 variants were constructed using the approach described inExample 3. The thermostability was determined as T½ using method I at68° C. as described in the “Materials & Methods” section and compared tothe wild-type A. niger AMG G2 under the same conditions. Evaluation ofvariants were performed on culture broth after filtration of thesupernatants. T½ A. niger AMG G2 T½ wild type Variant (min) (min) 25D357S + T360V + S371H 6.6 5.9 26 N313G + F318Y 8.9 5.9 27 S356P + S366T7.3 5.8 28 S340G + D357S + T360V + S386P 7.2 5.8

Example 9

[0990] Thermostability at 70° C.

[0991] An MG G2 variants was constructed using the approach described inExample 1 and evaluated as semi-purified (filtration of culture brothfollowed by desalting on a G-25 column) samples.

[0992] The thermostability was determined as % residual activity usingMethod I in 50 mM NaOAc, pH, 4.5, 70° C., as described in the “Material& Methods” section above. The result of the test is listed in the Tablebelow and compared to the wild-type A. niger AMG G2. Enzyme T½ (min) 29AMG G2 (wild type)  7 30 Y402F + S411V  60 31 S119P + Y402F + S411V 11532 S119P + Y312Q + Y402F + T416H  50

Example 10

[0993] thermostability at 70° C. in presence of 30% glucose

[0994] AMG G2 variants were constructed using the approach described inExample 3.

[0995] The thermostability was determined as T½ using method II at 70°C. as described in the “Materials & Methods” section and compared to thewild-type A. niger AMG G2 under the same conditions. Enzyme T½ (hr) 33AMG G2 (wild type) 1.5 34 Y402F 2.5 35 A393R 4.0 36 T2R + S386R + A393R2.0 37 PLASD(N-terminal) + V59A + A393R + T490A 16.0 

Example 11

[0996] Saccharification Performance of AMG Variants S119P+Y402F+S411Vand PLASD(N-terminal)+V59A+A393R+T490A, Respectively.

[0997] Saccharification performance of the AMG variantsS119P+Y402F+S411V and PLASD(N-terminal)+V59A+A393R+T490A, respectively,both having improved thermostability are tested at 70° C. as describedbelow.

[0998] Reference enzyme is the wild-type A. niger AMG G2.Saccharification is run under the following conditions: Substrate 10 DEMaltodextrin, approx. 30% DS (w/w) Temperature 70° C. Initial pH 4.3 (at70° C.) Enzyme dosage 0.24 AGU/g DS

[0999] Saccharification

[1000] The substrate for saccharification is made by dissolvingmaltodextrin (prepared from common corn) in boiling Milli-Q water andadjusting the dry substance to approximately 30% (w/w). pH is adjustedto 4.3. Aliquots of substrate corresponding to 15 g dry solids aretransferred to 50 ml blue cap glass flasks and placed in a water bathwith stirring. Enzymes are added and pH re-adjusted if necessary. Theexperiment is run in duplicate. Samples are taken periodically andanalysed at HPLC for determination of the carbohydrate composition.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 81 <210> SEQ ID NO 1<211> LENGTH: 1605 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger<220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION: (1)...(72)<221> NAME/KEY: mat_peptide <222> LOCATION: (73)...(1602) <221>NAME/KEY: CDS <222> LOCATION: (1)...(1602) <400> SEQUENCE: 1 atg tcg ttccga tct cta ctc gcc ctg agc ggc ctc gtc tgc aca ggg 48 Met Ser Phe ArgSer Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly -20 -15 -10 ttg gca aatgtg att tcc aag cgc gcg acc ttg gat tca tgg ttg agc 96 Leu Ala Asn ValIle Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser -5 1 5 aac gaa gcg accgtg gct cgt act gcc atc ctg aat aac atc ggg gcg 144 Asn Glu Ala Thr ValAla Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala 10 15 20 gac ggt gct tgg gtgtcg ggc gcg gac tct ggc att gtc gtt gct agt 192 Asp Gly Ala Trp Val SerGly Ala Asp Ser Gly Ile Val Val Ala Ser 25 30 35 40 ccc agc acg gat aacccg gac tac ttc tac acc tgg act cgc gac tct 240 Pro Ser Thr Asp Asn ProAsp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser 45 50 55 ggt ctc gtc ctc aag accctc gtc gat ctc ttc cga aat gga gat acc 288 Gly Leu Val Leu Lys Thr LeuVal Asp Leu Phe Arg Asn Gly Asp Thr 60 65 70 agt ctc ctc tcc acc att gagaac tac atc tcc gcc cag gca att gtc 336 Ser Leu Leu Ser Thr Ile Glu AsnTyr Ile Ser Ala Gln Ala Ile Val 75 80 85 cag ggt atc agt aac ccc tct ggtgat ctg tcc agc ggc gct ggt ctc 384 Gln Gly Ile Ser Asn Pro Ser Gly AspLeu Ser Ser Gly Ala Gly Leu 90 95 100 ggt gaa ccc aag ttc aat gtc gatgag act gcc tac act ggt tct tgg 432 Gly Glu Pro Lys Phe Asn Val Asp GluThr Ala Tyr Thr Gly Ser Trp 105 110 115 120 gga cgg ccg cag cga gat ggtccg gct ctg aga gca act gct atg atc 480 Gly Arg Pro Gln Arg Asp Gly ProAla Leu Arg Ala Thr Ala Met Ile 125 130 135 ggc ttc ggg cag tgg ctg cttgac aat ggc tac acc agc acc gca acg 528 Gly Phe Gly Gln Trp Leu Leu AspAsn Gly Tyr Thr Ser Thr Ala Thr 140 145 150 gac att gtt tgg ccc ctc gttagg aac gac ctg tcg tat gtg gct caa 576 Asp Ile Val Trp Pro Leu Val ArgAsn Asp Leu Ser Tyr Val Ala Gln 155 160 165 tac tgg aac cag aca gga tatgat ctc tgg gaa gaa gtc aat ggc tcg 624 Tyr Trp Asn Gln Thr Gly Tyr AspLeu Trp Glu Glu Val Asn Gly Ser 170 175 180 tct ttc ttt acg att gct gtgcaa cac cgc gcc ctt gtc gaa ggt agt 672 Ser Phe Phe Thr Ile Ala Val GlnHis Arg Ala Leu Val Glu Gly Ser 185 190 195 200 gcc ttc gcg acg gcc gtcggc tcg tcc tgc tcc tgg tgt gat tct cag 720 Ala Phe Ala Thr Ala Val GlySer Ser Cys Ser Trp Cys Asp Ser Gln 205 210 215 gca ccc gaa att ctc tgctac ctg cag tcc ttc tgg acc ggc agc ttc 768 Ala Pro Glu Ile Leu Cys TyrLeu Gln Ser Phe Trp Thr Gly Ser Phe 220 225 230 att ctg gcc aac ttc gatagc agc cgt tcc ggc aag gac gca aac acc 816 Ile Leu Ala Asn Phe Asp SerSer Arg Ser Gly Lys Asp Ala Asn Thr 235 240 245 ctc ctg gga agc atc cacacc ttt gat cct gag gcc gca tgc gac gac 864 Leu Leu Gly Ser Ile His ThrPhe Asp Pro Glu Ala Ala Cys Asp Asp 250 255 260 tcc acc ttc cag ccc tgctcc ccg cgc gcg ctc gcc aac cac aag gag 912 Ser Thr Phe Gln Pro Cys SerPro Arg Ala Leu Ala Asn His Lys Glu 265 270 275 280 gtt gta gac tct ttccgc tca atc tat acc ctc aac gat ggt ctc agt 960 Val Val Asp Ser Phe ArgSer Ile Tyr Thr Leu Asn Asp Gly Leu Ser 285 290 295 gac agc gag gct gttgcg gtg ggt cgg tac cct gag gac acg tac tac 1008 Asp Ser Glu Ala Val AlaVal Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr 300 305 310 aac ggc aac ccg tggttc ctg tgc acc ttg gct gcc gca gag cag ttg 1056 Asn Gly Asn Pro Trp PheLeu Cys Thr Leu Ala Ala Ala Glu Gln Leu 315 320 325 tac gat gct cta taccag tgg gac aag cag ggg tcg ttg gag gtc aca 1104 Tyr Asp Ala Leu Tyr GlnTrp Asp Lys Gln Gly Ser Leu Glu Val Thr 330 335 340 gat gtg tcg ctg gacttc ttc aag gca ctg tac agc gat gct gct act 1152 Asp Val Ser Leu Asp PhePhe Lys Ala Leu Tyr Ser Asp Ala Ala Thr 345 350 355 360 ggc acc tac tcttcg tcc agt tcg act tat agt agc att gta gat gcc 1200 Gly Thr Tyr Ser SerSer Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala 365 370 375 gtg aag act ttcgcc gat ggc ttc gtc tct att gtg gaa act cac gcc 1248 Val Lys Thr Phe AlaAsp Gly Phe Val Ser Ile Val Glu Thr His Ala 380 385 390 gca agc aac ggctcc atg tcc gag caa tac gac aag tct gat ggc gag 1296 Ala Ser Asn Gly SerMet Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu 395 400 405 cag ctt tcc gctcgc gac ctg acc tgg tct tat gct gct ctg ctg acc 1344 Gln Leu Ser Ala ArgAsp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr 410 415 420 gcc aac aac cgtcgt aac tcc gtc gtg cct gct tct tgg ggc gag acc 1392 Ala Asn Asn Arg ArgAsn Ser Val Val Pro Ala Ser Trp Gly Glu Thr 425 430 435 440 tct gcc agcagc gtg ccc ggc acc tgt gcg gcc aca tct gcc att ggt 1440 Ser Ala Ser SerVal Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly 445 450 455 acc tac agcagt gtg act gtc acc tcg tgg ccg agt atc gtg gct act 1488 Thr Tyr Ser SerVal Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr 460 465 470 ggc ggc accact acg acg gct acc ccc act gga tcc ggc agc gtg acc 1536 Gly Gly Thr ThrThr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr 475 480 485 tcg acc agcaag acc acc gcg act gct agc aag acc agc acc acg acc 1584 Ser Thr Ser LysThr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr 490 495 500 cgc tct ggtatg tca ctg tga 1605 Arg Ser Gly Met Ser Leu 505 510 <210> SEQ ID NO 2<211> LENGTH: 534 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger<220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(24) <400>SEQUENCE: 2 Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys ThrGly -20 -15 -10 Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser TrpLeu Ser -5 1 5 Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn IleGly Ala 10 15 20 Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val ValAla Ser 25 30 35 40 Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp ThrArg Asp Ser 45 50 55 Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg AsnGly Asp Thr 60 65 70 Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala GlnAla Ile Val 75 80 85 Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser GlyAla Gly Leu 90 95 100 Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala TyrThr Gly Ser Trp 105 110 115 120 Gly Arg Pro Gln Arg Asp Gly Pro Ala LeuArg Ala Thr Ala Met Ile 125 130 135 Gly Phe Gly Gln Trp Leu Leu Asp AsnGly Tyr Thr Ser Thr Ala Thr 140 145 150 Asp Ile Val Trp Pro Leu Val ArgAsn Asp Leu Ser Tyr Val Ala Gln 155 160 165 Tyr Trp Asn Gln Thr Gly TyrAsp Leu Trp Glu Glu Val Asn Gly Ser 170 175 180 Ser Phe Phe Thr Ile AlaVal Gln His Arg Ala Leu Val Glu Gly Ser 185 190 195 200 Ala Phe Ala ThrAla Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln 205 210 215 Ala Pro GluIle Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe 220 225 230 Ile LeuAla Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr 235 240 245 LeuLeu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp 250 255 260Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu 265 270275 280 Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser285 290 295 Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr TyrTyr 300 305 310 Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala GluGln Leu 315 320 325 Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser LeuGlu Val Thr 330 335 340 Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr SerAsp Ala Ala Thr 345 350 355 360 Gly Thr Tyr Ser Ser Ser Ser Ser Thr TyrSer Ser Ile Val Asp Ala 365 370 375 Val Lys Thr Phe Ala Asp Gly Phe ValSer Ile Val Glu Thr His Ala 380 385 390 Ala Ser Asn Gly Ser Met Ser GluGln Tyr Asp Lys Ser Asp Gly Glu 395 400 405 Gln Leu Ser Ala Arg Asp LeuThr Trp Ser Tyr Ala Ala Leu Leu Thr 410 415 420 Ala Asn Asn Arg Arg AsnSer Val Val Pro Ala Ser Trp Gly Glu Thr 425 430 435 440 Ser Ala Ser SerVal Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly 445 450 455 Thr Tyr SerSer Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr 460 465 470 Gly GlyThr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr 475 480 485 SerThr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr 490 495 500Arg Ser Gly Met Ser Leu 505 510 <210> SEQ ID NO 3 <211> LENGTH: 30 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer 7258 <400> SEQUENCE: 3 gaatgacttg gttgacgcgtcaccagtcac 30 <210> SEQ ID NO 4 <211> LENGTH: 68 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer 21401 <400> SEQUENCE: 4 ggggatcatg ataggactag ccatattaatgaagggcata taccacgcct tggacctgcg 60 ttatagcc 68 <210> SEQ ID NO 5 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer 107581 <400> SEQUENCE: 5gcaacgaagc gcccgtggct cgtac 25 <210> SEQ ID NO 6 <211> LENGTH: 88 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer FAMGIL <400> SEQUENCE: 6 cgaagcgacc gtggctcgtactgccatcta taacatcggc gcgtctgtgc gcggtggcat 60 tgtcgttgct agtcccagcacggataac 88 <210> SEQ ID NO 7 <211> LENGTH: 28 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Primer RAMG1 <400> SEQUENCE: 7 gatggcagta cgagccacgg tcgcttcg 28 <210>SEQ ID NO 8 <211> LENGTH: 75 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PRIMER FAMGIV <400>SEQUENCE: 8 gtgtcgctgg acttcttcaa gaacctctta ccctactaca gtcgttatcattgatgccgt 60 gaagactttc gccga 75 <210> SEQ ID NO 9 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: PRIMER RAMGVI <400> SEQUENCE: 9cttgaagaag tccagcgaca c 21 <210> SEQ ID NO 10 <211> LENGTH: 27 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer FG2 <400> SEQUENCE: 10 catccccagg atccttactc agcaatg27 <210> SEQ ID NO 11 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer RG2<400> SEQUENCE: 11 ctcaaacgac tcaccagcct ctagagt 27 <210> SEQ ID NO 12<211> LENGTH: 2602 <212> TYPE: DNA <213> ORGANISM: ASPERGILLUS NIGER<400> SEQUENCE: 12 ttcgtcgcct aatgtctcgt ccgttcacaa actgaagagcttgaagtggc gagatgtctc 60 tgcaggaatt caagctagat gctaagcgat attgcatggcaatatgtgtt gatgcatgtg 120 cttcttcctt cagcttcccc tcgtgcgagt gaggtttggctataaattga agtggttggt 180 cggggttccg tgaggggctg aagtgcttcc tcccttttaggcgcaactga gagcctgagc 240 ttcatcccca gcatcattac acctcagcaa tgtcgttccgatctctactc gccctgagcg 300 gcctcgtctg cacagggttg gcaaatgtga tttccaagcgcgcgaccttg gattcatggt 360 tgagcaacga agcgaccgtg gctcgtactg ccatcctgaataacatcggg gcggacggtg 420 cttgggtgtc gggcgcggac tctggcattg tcgttgctagtcccagcacg gataacccgg 480 actgtatgtt tcgagctcag atttagtatg agtgtgtcattgattgattg atgctgactg 540 gcgtgtcgtt tgttgtagac ttctacacct ggactcgcgactctggtctc gtcctcaaga 600 ccctcgtcga tctcttccga aatggagata ccagtctcctctccaccatt gagaactaca 660 tctccgccca ggcaattgtc cagggtatca gtaacccctctggtgatctg tccagcggcg 720 ctggtctcgg tgaacccaag ttcaatgtcg atgagactgcctacactggt tcttggggac 780 ggccgcagcg agatggtccg gctctgagag caactgctatgatcggcttc gggcagtggc 840 tgcttgtatg ttctccaccc ccttgcgtct gatctgtgacatatgtagct gactggtcag 900 gacaatggct acaccagcac cgcaacggac attgtttggcccctcgttag gaacgacctg 960 tcgtatgtgg ctcaatactg gaaccagaca ggatatggtgtgtttgtttt attttaaatt 1020 tccaaagatg cgccagcaga gctaacccgc gatcgcagatctctgggaag aagtcaatgg 1080 ctcgtctttc tttacgattg ctgtgcaaca ccgcgcccttgtcgaaggta gtgccttcgc 1140 gacggccgtc ggctcgtcct gctcctggtg tgattctcaggcacccgaaa ttctctgcta 1200 cctgcagtcc ttctggaccg gcagcttcat tctggccaacttcgatagca gccgttccgg 1260 caaggacgca aacaccctcc tgggaagcat ccacacctttgatcctgagg ccgcatgcga 1320 cgactccacc ttccagccct gctccccgcg cgcgctcgccaaccacaagg aggttgtaga 1380 ctctttccgc tcaatctata ccctcaacga tggtctcagtgacagcgagg ctgttgcggt 1440 gggtcggtac cctgaggaca cgtactacaa cggcaacccgtggttcctgt gcaccttggc 1500 tgccgcagag cagttgtacg atgctctata ccagtgggacaagcaggggt cgttggaggt 1560 cacagatgtg tcgctggact tcttcaaggc actgtacagcgatgctgcta ctggcaccta 1620 ctcttcgtcc agttcgactt atagtagcat tgtagatgccgtgaagactt tcgccgatgg 1680 cttcgtctct attgtggtaa gtctacgcta gacaagcgctcatgttgaca gagggtgcgt 1740 actaacagaa gtaggaaact cacgccgcaa gcaacggctccatgtccgag caatacgaca 1800 agtctgatgg cgagcagctt tccgctcgcg acctgacctggtcttatgct gctctgctga 1860 ccgccaacaa ccgtcgtaac tccgtcgtgc ctgcttcttggggcgagacc tctgccagca 1920 gcgtgcccgg cacctgtgcg gccacatctg ccattggtacctacagcagt gtgactgtca 1980 cctcgtggcc gagtatcgtg gctactggcg gcaccactacgacggctacc cccactggat 2040 ccggcagcgt gacctcgacc agcaagacca ccgcgactgctagcaagacc agcaccagta 2100 cgtcatcaac ctcctgtacc actcccaccg ccgtggctgtgactttcgat ctgacagcta 2160 ccaccaccta cggcgagaac atctacctgg tcggatcgatctctcagctg ggtgactggg 2220 aaaccagcga cggcatagct ctgagtgctg acaagtacacttccagcgac ccgctctggt 2280 atgtcactgt gactctgccg gctggtgagt cgtttgagtacaagtttatc cgcattgaga 2340 gcgatgactc cgtggagtgg gagagtgatc ccaaccgagaatacaccgtt cctcaggcgt 2400 gcggaacgtc gaccgcgacg gtgactgaca cctggcggtgacaatcaatc catttcgcta 2460 tagttaaagg atggggatga gggcaattgg ttatatgatcatgtatgtag tgggtgtgca 2520 taatagtagt gaaatggaag ccaagtcatg tgattgtaatcgaccgacgg aattgaggat 2580 atccggaaat acagacaccg gg 2602 <210> SEQ ID NO13 <211> LENGTH: 640 <212> TYPE: PRT <213> ORGANISM: ASPERGILLUS NIGER<400> SEQUENCE: 13 Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu ValCys Thr Gly 1 5 10 15 Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu AspSer Trp Leu Ser 20 25 30 Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu AsnAsn Ile Gly Ala 35 40 45 Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly IleVal Val Ala Ser 50 55 60 Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr TrpThr Arg Asp Ser 65 70 75 80 Gly Leu Val Leu Lys Thr Leu Val Asp Leu PheArg Asn Gly Asp Thr 85 90 95 Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile SerAla Gln Ala Ile Val 100 105 110 Gln Gly Ile Ser Asn Pro Ser Gly Asp LeuSer Ser Gly Ala Gly Leu 115 120 125 Gly Glu Pro Lys Phe Asn Val Asp GluThr Ala Tyr Thr Gly Ser Trp 130 135 140 Gly Arg Pro Gln Arg Asp Gly ProAla Leu Arg Ala Thr Ala Met Ile 145 150 155 160 Gly Phe Gly Gln Trp LeuLeu Asp Asn Gly Tyr Thr Ser Thr Ala Thr 165 170 175 Asp Ile Val Trp ProLeu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln 180 185 190 Tyr Trp Asn GlnThr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser 195 200 205 Ser Phe PheThr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser 210 215 220 Ala PheAla Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln 225 230 235 240Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe 245 250255 Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr 260265 270 Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp275 280 285 Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His LysGlu 290 295 300 Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp GlyLeu Ser 305 310 315 320 Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro GluAsp Thr Tyr Tyr 325 330 335 Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu AlaAla Ala Glu Gln Leu 340 345 350 Tyr Asp Ala Leu Tyr Gln Trp Asp Lys GlnGly Ser Leu Glu Val Thr 355 360 365 Asp Val Ser Leu Asp Phe Phe Lys AlaLeu Tyr Ser Asp Ala Ala Thr 370 375 380 Gly Thr Tyr Ser Ser Ser Ser SerThr Tyr Ser Ser Ile Val Asp Ala 385 390 395 400 Val Lys Thr Phe Ala AspGly Phe Val Ser Ile Val Glu Thr His Ala 405 410 415 Ala Ser Asn Gly SerMet Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu 420 425 430 Gln Leu Ser AlaArg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr 435 440 445 Ala Asn AsnArg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr 450 455 460 Ser AlaSer Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly 465 470 475 480Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr 485 490495 Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr 500505 510 Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser Thr515 520 525 Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala Val Ala Val Thr PheAsp 530 535 540 Leu Thr Ala Thr Thr Thr Tyr Gly Glu Asn Ile Tyr Leu ValGly Ser 545 550 555 560 Ile Ser Gln Leu Gly Asp Trp Glu Thr Ser Asp GlyIle Ala Leu Ser 565 570 575 Ala Asp Lys Tyr Thr Ser Ser Asp Pro Leu TrpTyr Val Thr Val Thr 580 585 590 Leu Pro Ala Gly Glu Ser Phe Glu Tyr LysPhe Ile Arg Ile Glu Ser 595 600 605 Asp Asp Ser Val Glu Trp Glu Ser AspPro Asn Arg Glu Tyr Thr Val 610 615 620 Pro Gln Ala Cys Gly Thr Ser ThrAla Thr Val Thr Asp Thr Trp Arg 625 630 635 640 <210> SEQ ID NO 14 <211>LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: primer K1-T2X n at positions 22 and 23is a or g or c or t <400> SEQUENCE: 14 atgtgatttc caagcgcgcg vnnttggattcatggttgag caa 43 <210> SEQ ID NO 15 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-N9X n at positions 22 and 23 is a or g or c or t<400> SEQUENCE: 15 ccttggattc atggttgagc vnngaagcga ccgtggctcg tac 43<210> SEQ ID NO 16 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-A11X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE: 16attcatggtt gagcaacgaa vnnaccgtgg ctcgtactgc cat 43 <210> SEQ ID NO 17<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-L66X n at positions22 and 23 is a or g or c or t <400> SEQUENCE: 17 tcctcaagac cctcgtcgatvnnttccgaa atggagatac cag 43 <210> SEQ ID NO 18 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-S386X n at positions 22 and 23 is a or g or c ort <400> SEQUENCE: 18 ctttcgccga tggcttcgtc vnnattgtgg aaactcacgc cgc 43<210> SEQ ID NO 19 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-E389X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE:19 atggcttcgt ctctattgtg vnnactcacg ccgcaagcaa cgg 43 <210> SEQ ID NO 20<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-T390X n at positions22 and 23 are a or g or c <400> SEQUENCE: 20 gcttcgtctc tattgtggaavnncacgccg caagcaacgg ctc 43 <210> SEQ ID NO 21 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-A393X n at positions 22 and 23 is a or g or c ort <400> SEQUENCE: 21 ctattgtgga aactcacgcc vnnagcaacg gctccatgtc cga 43<210> SEQ ID NO 22 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-S394X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE:22 ttgtggaaac tcacgccgca vnnaacggct ccatgtccga gca 43 <210> SEQ ID NO 23<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-395X n at positions22 and 23 is a or g or c or t <400> SEQUENCE: 23 tggaaactca cgccgcaagcvnnggctcca tgtccgagca ata 43 <210> SEQ ID NO 24 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-G396X n at positions 22 and 23 is a or g or c ort <400> SEQUENCE: 24 aaactcacgc cgcaagcaac vnntccatgt ccgagcaata cga 43<210> SEQ ID NO 25 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-K404X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE:25 ccatgtccga gcaatacgac vnntctgatg gcgagcagct ttc 43 <210> SEQ ID NO 26<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-D406X n at positions22 and 23 is a or g or c or t <400> SEQUENCE: 26 ccgagcaata cgacaagtctvnnggcgagc agctttccgc tcg 43 <210> SEQ ID NO 27 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-E408X n at positions 22 and 23 is a or g or c ort <400> SEQUENCE: 27 aatacgacaa gtctgatggc vnncagcttt ccgctcgcga cct 43<210> SEQ ID NO 28 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-L410X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE:28 acaagtctga tggcgagcag vnntccgctc gcgacctgac ct 42 <210> SEQ ID NO 29<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-L423X n at positions22 and 23 is a or g or c or t <400> SEQUENCE: 29 cctggtctta tgctgctctgvnnaccgcca acaaccgtcg taa 43 <210> SEQ ID NO 30 <211> LENGTH: 44 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk1-N426X n at positions 22 and 23 is a or g or c ort <400> SEQUENCE: 30 atgctgctct gctgaccgcc vnnaaccgtc gtaactccgt cgtg 44<210> SEQ ID NO 31 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk1-N427X n at positions 22 and 23 is a or g or c or t <400> SEQUENCE:31 ctgctctgct gaccgccaac vnncgtcgta actccgtcgt gcct 44 <210> SEQ ID NO32 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk1-Y402X n at positions21 and 22 is a or g or c or t <400> SEQUENCE: 32 acggctccat gtccgagcaanncgacaagt ctgatggcga gcagct 46 <210> SEQ ID NO 33 <211> LENGTH: 41<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: primer Hk2-L234X-sense n at positions 20 and 21 is aor g or c or t <400> SEQUENCE: 33 ctggaccggc agcttcattn nkgccaacttcgatagcagc c 41 <210> SEQ ID NO 34 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-A235S-antisense <400> SEQUENCE: 34 gaacggctgctatcgaagtt agacagaatg aagctgccgg tc 42 <210> SEQ ID NO 35 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-NF237X-sense n at position 20 is aor g or c or t <400> SEQUENCE: 35 cagcttcatt ctggccaacn atgatagcagccgttccggc a 41 <210> SEQ ID NO 36 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-D235T-antisense <400> SEQUENCE: 36 ccttgccggaacggctgcta gtgaagttgg ccagaatgaa gc 42 <210> SEQ ID NO 37 <211> LENGTH:42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-D238S-antisense <400> SEQUENCE: 37ccttgccgga acggctgcta gagaagttgg ccagaatgaa gc 42 <210> SEQ ID NO 38<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk2-S239X-sense n atpositions 21 and 22 is a or g or c or t <400> SEQUENCE: 38 tcattctggccaacttcgat nncagccgtt ccggcaagga cg 42 <210> SEQ ID NO 39 <211> LENGTH:42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-S240G-antisense <400> SEQUENCE: 39ttgcgtcctt gccggaacga ccgctatcga agttggccag aa 42 <210> SEQ ID NO 40<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk2-S242X-antisense n atposition 22 is a or g or c or t <400> SEQUENCE: 40 gggtgtttgc gtccttgccaknacggctgc tatcgaagtt g 41 <210> SEQ ID NO 41 <211> LENGTH: 41 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-G243X-antisense n at position 22 is a or g or cor t <400> SEQUENCE: 41 ggagggtgtt tgcgtcctta knggaacggc tgctatcgaa g 41<210> SEQ ID NO 42 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk2-K244R-sense <400> SEQUENCE: 42 cgatagcagc cgttccggca gagacgcaaacaccctcctg g 41 <210> SEQ ID NO 43 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-T310V-antisense <400> SEQUENCE: 43 acgggttgccgttgtagtaa acgtcctcag ggtaccgacc c 41 <210> SEQ ID NO 44 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-T310S-antisense <400> SEQUENCE: 44acgggttgcc gttgtagtaa gagtcctcag ggtaccgacc c 41 <210> SEQ ID NO 45<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk2-Y311N-sense <400>SEQUENCE: 45 tcggtaccct gaggacacga attacaacgg caacccgtgg t 41 <210> SEQID NO 46 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Hk2-Y312Q-antisense<400> SEQUENCE: 46 ggaaccacgg gttgccgttt tggtacgtgt cctcagggta c 41<210> SEQ ID NO 47 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk2-Y312N-antisense <400> SEQUENCE: 47 ggaaccacgg gttgccgtta ttgtacgtgtcctcagggta c 41 <210> SEQ ID NO 48 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-N313T-sense <400> SEQUENCE: 48 ccctgaggacacgtactaca ctggcaaccc gtggttcctg t 41 <210> SEQ ID NO 49 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-N313S-sense <400> SEQUENCE: 49ccctgaggac acgtactact ctggcaaccc gtggttcctg t 41 <210> SEQ ID NO 50<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk2-N313G-sense <400>SEQUENCE: 50 ccctgaggac acgtactacg gtggcaaccc gtggttcctg t 41 <210> SEQID NO 51 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primerHk2-N315Q-antisense <400> SEQUENCE: 51 aggtgcacag gaaccacggt tggccgttgtagtacgtgtc c 41 <210> SEQ ID NO 52 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk2-N315E-antisense <400> SEQUENCE: 52 aggtgcacaggaaccacggt tcgccgttgt agtacgtgtc c 41 <210> SEQ ID NO 53 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk2-N315R-antisense <400> SEQUENCE: 53aggtgcacag gaaccacggt ctgccgttgt agtacgtgtc c 41 <210> SEQ ID NO 54<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk2-F318Y-antisense <400>SEQUENCE: 54 cggcagccaa ggtgcacaga taccacgggt tgccgttgta g 41 <210> SEQID NO 55 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer Hk2-Q409P-sense<400> SEQUENCE: 55 cgacaagtct gatggcgagc cactttccgc tcgcgacctg a 41<210> SEQ ID NO 56 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-D336X-sense n at positions 20 and 21 is a or g or c or t <400>SEQUENCE: 56 cgatgctcta taccagtggn nkaagcaggg gtcgttggag g 41 <210> SEQID NO 57 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer Hk3-K337X-sensen at positions 20 and 21 is a or g or c or t <400> SEQUENCE: 57tgctctatac cagtgggacn nkcaggggtc gttggaggtc a 41 <210> SEQ ID NO 58<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk3-Q338X-antisense n atpositions 21 and 22 is a or g or c or t <400> SEQUENCE: 58 ctgtgacctccaacgacccg nncttgtccc actggtatag a 41 <210> SEQ ID NO 59 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk3-G339X-sense n at position 20 is a org or c or t <400> SEQUENCE: 59 ataccagtgg gacaagcagn cutcgttggaggtcacagat g 41 <210> SEQ ID NO 60 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk3-S340X′-antisense n at position 21 is a or g or cor t <400> SEQUENCE: 60 acacatctgt gacctccaaa ntcccctgct tgtcccactg g 41<210> SEQ ID NO 61 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-S340X“-antisense n at position 21 is a or g or c or t <400>SEQUENCE: 61 acacatctgt gacctccaaa nccccctgct tgtcccactg g 41 <210> SEQID NO 62 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer Hk3-L341X-sensen at position 20 is a or g or c or t <400> SEQUENCE: 62 gtgggacaagcaggggtcgn uugaggtcac agatgtgtcg c 41 <210> SEQ ID NO 63 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk3-K352Q-sense <400> SEQUENCE: 63tgtgtcgctg gacttcttcc aagcactgta cagcgatgct g 41 <210> SEQ ID NO 64<211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk3-K352R-sense <400>SEQUENCE: 64 tgtgtcgctg gacttcttca gagcactgta cagcgatgct g 41 <210> SEQID NO 65 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-A352D-antisense <400> SEQUENCE: 65 tagcagcatc gctgtacaga tccttgaagaagtccagcga c 41 <210> SEQ ID NO 66 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer HK3-A353S-antisense <400> SEQUENCE: 66 tagcagcatcgctgtacaga gacttgaaga agtccagcga c 41 <210> SEQ ID NO 67 <211> LENGTH:42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk3-S356P-sense <400> SEQUENCE: 67acttcttcaa ggcactgtac ccagatgctg ctactggcac ct 42 <210> SEQ ID NO 68<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk3-S356N-sense <400>SEQUENCE: 68 acttcttcaa ggcactgtac aaugatgctg ctactggcac cta 43 <210>SEQ ID NO 69 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer Hk3-S356D-sense<400> SEQUENCE: 69 acttcttcaa ggcactgtac gaugatgctg ctactggcac cta 43<210> SEQ ID NO 70 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-D357S-antisense <400> SEQUENCE: 70 gagtaggtgc cagtagcagc agagctgtacagtgccttga aga 43 <210> SEQ ID NO 71 <211> LENGTH: 41 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk3-A359S-sense <400> SEQUENCE: 71 ggcactgtacagcgatgctt ctactggcac ctactcttcg t 41 <210> SEQ ID NO 72 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk3-T360V-antisense <400> SEQUENCE: 72tggacgaaga gtaggtgcca acagcagcat cgctgtacag t 41 <210> SEQ ID NO 73<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk3-G361X-sense n atposition 21 is a or g or c or t <400> SEQUENCE: 73 tgtacagcga tgctgctactnctacctact cttcgtccag ttc 43 <210> SEQ ID NO 74 <211> LENGTH: 42 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk3-T362R-antisense <400> SEQUENCE: 74 gtcgaactggacgaagagta tctgccagta gcagcatcgc tg 42 <210> SEQ ID NO 75 <211> LENGTH:42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: primer Hk3-S364X-sense n at positions 20 and 21is a or g or c or t <400> SEQUENCE: 75 tgctgctact ggcacctacn nktcgtccagttcgacttat ag 42 <210> SEQ ID NO 76 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk3-S365X-sense n at positions 20 and 21 is a or gor c or t <400> SEQUENCE: 76 tgctactggc acctactctn nktccagttc gacttatagtag 42 <210> SEQ ID NO 77 <211> LENGTH: 42 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:primer Hk3-S366T-antisense <400> SEQUENCE: 77 atgctactat aagtcgaactagtcgaagag taggtgccag ta 42 <210> SEQ ID NO 78 <211> LENGTH: 42 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer Hk3-S368X-antisense n at position 23 is a or g or cor t <400> SEQUENCE: 78 tctacaatgc tactataagt agnactggac gaagagtagg tg42 <210> SEQ ID NO 79 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-T369X-sense n at positions 20 and 21 is a or g or c or t <400>SEQUENCE: 79 ctactcttcg tccagttcgn nktatagtag cattgtagat gcc 43 <210>SEQ ID NO 80 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primerHk3-S371X-antisense n at position 23 is a or g or c or t <400> SEQUENCE:80 ttcacggcat ctacaatgct atnataagtc gaactggacg aag 43 <210> SEQ ID NO 81<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer Hk3-S372X-sense n atpositions 21 and 22 is a or g or c or t <400> SEQUENCE: 81 cgtccagttcgacttatagt nntattgtag atgccgtgaa gac 43

1. A variant of a parent glucoamylase comprising one or more mutation(s)in the following position(s) or region(s) in the amino acid sequenceshown in NO: 2: Region: 1-18, Region: 19-35, Region: 40-62, Region:73-80, Region: 93-127, Region: 170-184, Region: 200-212, Region:234-246, Region: 287-319, Region: 334-341, Region: 353-374, Region:388-414, Region: 445-470, and/or in a corresponding position or regionin a homologous glucoamylase which displays at least 60% homology withthe amino acid sequences shown in SEQ ID NO: 2, with the exception ofthe following substitutions: N20C, A27C, S30P, Y48W, Y50F, W52F, R54K/L,D55G/V, G57A, K108R, D112Y, Y116A/W, S119C/W/E/G/Y/P, W120H/L/F/Y,G121T/A, R122Y, P123G, Q124H, R125K, W170F, N171S, Q172N, T173G, G174C,Y175F, D176N/E, L177H/D, W178R/D, E179Q/D, E180D/Q, V181D/A/T,N182A/D/Q/Y/S, G183K, S184H, W212F, R241K, A246C, D293E/Q, A302V, R305K,Y306F, D309N/E, Y312W, W317F, E389D/Q, H391W, A392D, A393P, N395Q,G396S, E400Q/C, Q401E, G407D, E408P, L410F, S411A/G/C/H/D, S460P.
 2. Thevariant of claim 1, wherein the variant comprise one or more of thefollowing mutations: A1V, T2E/P/Q/R/H/M, L3P/N, N9A, A11P/E, I18V, L19N,N20T, G23A, A24S/T, D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V,D33R/K/H, S34N, S40C, T43R, T51D/S, T53D, S56A/C, V59T/A, L60A, N93T,P94V, S95N, D97S, L98P/S, S100T/D, A102S/*, N110T, V111P, D112N,E113M/A,T114S, A115Q/G, Y116F, S119A, G127A, N182E, A201D, F202L, A203L, T204K,A205R/S, V206L/N, G207N, S208H/T/D, S209T, S211P, W212N/A/T, A246TY312Q, N313T/S/G, A353D/S, S356P/N/D, D357S, A359S, T360V, G361S/P/T/A,T362R, S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S366T, S368P/T/A,T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S371Y/H/N/D,S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G, T390R, A393R, S394R/P, M398L,S399C/Q/T, Y402F, D403S, S405T, D406N,E408C/R, L410I/R, S411V, A412C,D414A, G447S, S465P.
 3. A variant of a parent glucoamylase with improvedthermostability comprising one or more mutation(s) in the followingposition(s) or region(s) in the amino acid sequence shown in NO: 2:Region: 1-18, Region: 19-35, Region: 73-80, Region: 93-127, Region:170-184, Region: 200-212, Region: 234-246, Region: 287-319 Region:334-341, Region: 353-374, Region: 388-414. Region: 445-470, and/or in acorresponding position or region in a homologous glucoamylase whichdisplays at least 60% homology with the amino acid sequences shown inSEQ ID NO: 2, with the exception of the following substitutions: N20C,A27C, S30P, A246C.
 4. A variant of a parent glucoamylase with increasedspecific activity comprising one or more mutation(s) in the followingposition(s) or region(s) in the amino acid sequence shown in NO: 2:Region: 1-18, Region: 40-62, Region: 93-127, Region: 170-184, Region:200-212, Region: 234-246, Region: 287-319, Region: 388-414, and/or in acorresponding position or region in a homologous glucoamylase whichdisplays at least 60% homology with the amino acid sequences shown inSEQ ID NO: 2, with the exception of the following substitutions: S411G.5. The variant according to claim 4, having one or more mutation(s) inthe following region(s) in the amino acid sequence shown in NO: 2:Region: 287-300, Region: 305-319, and/or in a corresponding position orregions in a homologous glucoamylase which displays at least 60%homology with the amino acid sequences shown in SEQ ID NO:
 2. 6. Thevariant according to any of claims 1-5, wherein the parent homologousglucoamylase is the Aspergillus niger G1 glucoamylase.
 7. The variantaccording to any of claims 1-6, wherein the glucoamylase is a truncatedglucoamylase, in particular in the C-terminal.
 8. A DNA constructcomprising a DNA sequence encoding a glucoamylase variant according toany one of claims 1-7.
 9. A recombinant expression vector which carriesa DNA construct according to claim
 8. 10. A cell which is transformedwith a DNA construct according to claim 8 or a vector according to claim9.
 11. A cell according to claim 10, which is a microorganism, such as abacterium or a fungus.
 12. The cell according to claim 11, which is aprotease deficient Aspergillus oryzae or Aspergillus niger.
 13. Aprocess for converting starch or partially hydrolyzed starch into asyrup containing dextrose, said process including the step saccharifyingstarch hydrolyzate in the presence of a glucoamylase variant accordingto any of claims 1-7.
 14. The process of claim 14, wherein the dosage ofglucoamylase is present in the range from 0.05 to 0.5 AGU per gram ofdry solids.
 15. The process of any claims 13 or 14, comprisingsaccharification of a starch hydrolyzate of at least 30 percent byweight of dry solids.
 16. The process of any of the preceding claims,wherein the saccharification is conducted in the presence of adebranching enzyme selected from the group of pullulanase andisoamylase, preferably a pullulanase derived from Bacillusacidopullulyticus or Bacillus deramificans or an isoamylase derived fromPseudomonas amyloderamosa.
 17. The process of any of the precedingclaims, wherein the saccharification is conducted at a pH of 3 to 5.5and at a temperature of 60-80° C., preferably 63-75° C., for 24 to 72hours, preferably for 36-48 hours at a pH from 4 to 4.5.
 18. A method ofsaccharifying a liquefied starch solution, which method comprises (i) asaccharification step during which step one or more enzymaticsaccharification stages takes place, and the subsequent step of (ii) oneor more high temperature membrane separation steps wherein the enzymaticsaccharification is carried out using a glucoamylase variant accordingto any of claim 1 to
 7. 19. Use of a glucoamylase variant according toany of claims 1-7 in a starch conversion process.
 20. Use of aglucoamylase variant according to any of claims 1-7 in a continuousstarch conversion process.
 21. Use according to claim 20, wherein thecontinuous starch conversion process include a continuoussaccharification process according to claim
 18. 22. Use of aglucoamylase variant according to any of claims 1-7 in a process forproducing oligosaccharides.
 23. Use of a glucoamylase variant accordingto any of claims 1-7 in a process for producing specialty syrups. 24.Use of a glucoamylase variant according to any one of claims 1-7 in aprocess for producing ethanol for fuel.
 25. Use of a glucoamylasevariant according to any one of claims 1-7 in a process for producing abeverage.
 26. Use of a glucoamylase variant according to any one ofclaims 1-7 in a fermentation process for producing organic compounds,such as citric acid, ascorbic acid, lysine, glutamic acid.
 27. A methodfor improving the thermostability and/or of increasing the specificactivity of a parent glucoamylase by making a mutation in one or more ofthe following position(s) or region(s) in the amino acid sequence shownin NO: 2: Region: 1-18, Region: 19-35, Region: 40-62, Region: 73-80,Region: 93-127, Region: 170-184, Region: 200-212, Region: 234-246,Region: 287-319, Region: 334-341, Region: 353-374, Region: 388-414,Region: 445-470, and/or in a corresponding position or region in ahomologous glucoamylase which displays at least 60% homology with theamino acid sequences shown in SEQ ID NO:
 2. 28. The method according toclaim 27, having one or more of the following substitutions: A1V,T2E/P/Q/R/H/M, L3P/N, N9A, A11P/E, I18V, L19N, N20T, G23A, A24S/T,D25S/T/R, G26A, A27S/T, W28R/Y, S30T/N, G31A, A32V, D33R/K/H, S34N,S40C, T43R, T51D/S, T53D, S56A/C, V59T/A, L60A, N93T, P94V, S95N, D97S,L98P/S, S100T/D, A102S/*, N110T, V111P, D112N,E113M/A, T114S, A115Q/G,Y116F, S119A, G127A, N182E, A201D, F202L, A203L, T204K, A205R/S,V206L/N, G207N, S208H/T/D, S209T, S211P, W212N/A/T, A246T Y312Q,N313T/S/G, A353D/S, S356P/N/D, D357S, A359S, T360V, G361S/P/T/A, T362R,S364A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V,S365A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S366T, S368P/T/A,T369A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/T/W/Y/V, S371Y/H/N/D,S372F/Y/C/L/P/H/R/I/T/N/S/V/A/D/G, T390R, A393R, S394R/P, M398L,S399C/Q/T, Y402F, D403S, S405T, D406N,E408C/R, L410I/R, S411V, A412C,D414A, G447S, S465P.